Aptamers for clostridium difficile diagnostics

ABSTRACT

The present disclosure relates generally to the field of nucleic acids and, more particularly, to aptamers capable of binding to toxins produced by  Clostridium difficile ; diagnostic kits and methods comprising such aptamers; and methods of making and using such aptamers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Ser. No. 61/451,227, which was filed on Mar. 10, 2011, the disclosure of which is incorporated herein in its entirety.

FIELD OF THE INVENTION

The present disclosure relates generally to the field of nucleic acids and more particularly to aptamers capable of binding to toxins produced by C. difficile and useful for diagnostic tests for C. difficile. The disclosure further relates to materials and methods for diagnosing C. difficile contamination or infection.

Incorporated by reference herein in its entirety is the Sequence Listing entitled “20120221SequenceListing005741_ST25.txt”, created Mar. 9, 2012, size of 9 kilobytes.

BACKGROUND

The following description provides a summary of information relevant to the present disclosure and is not an admission that any of the information provided or publications referenced herein is prior art to the present disclosure.

C. difficile infection (CDI) has been on the rise worldwide over the last several years. The clinical and economic consequences are substantial, with more than half a million cases and estimated costs of 3.2 billion dollars per year for CDI management in the U.S. alone (O'Brien, J. A., et al., Infect. Control Hosp. Epidemiol., 2007. 28(11): p. 1219-27).

CDI is an inflammatory condition of the large bowel characterized by diarrhea and can range in severity from mild to fulminant. More severe CDI syndromes are pseudomembraneous colitis and toxic megacolon. Most CDI cases occur in elderly patients in a hospital setting or in nursing homes. Hospitalization, however, increases the risk of colonization also for healthy adults. In the U.S., CDI hospitalizations and CDI-related case-fatality rates doubled between 2000 and 2005. A number of recent outbreaks have been reported in which CDI cases were primarily clonal in nature. A strain type classified as BI/NAP1/027 was responsible for more than half of the cases, and hallmarks of this epidemic “outbreak” strain are high morbidity and mortality, higher resistance to antibiotics (e.g. fluoroquinolones), the presence of a tcdC variant gene, and toxin hyper-production (Freeman, J., et al., Clin. Microbiol. Rev., 2010. 23(3): p. 529-49; Rupnik, M., M. H. Wilcox, and D. N. Gerding, Nat. Rev. Microbiol., 2009. 7(7): p. 526-36).

Use of antibiotics is a strong predisposing factor for CDI due to the disruption of the normal gut flora that otherwise suppresses C. difficile. Ingestion of spores is the main route of colonization of the human gut by C. difficile. Spores are extremely resistant to disinfectants and can persist in the environment for more than 12 months with little loss of viability or pathogenicity. Spores are also implicated in the 20-25% of CDI cases which relapse after treatment. Current treatment regimens for CDI are vancomycin or metronidazole. Several new, more selective agents that hold promise to reduce CDI recurrence rates are in clinical development.

The inflammation of the intestinal lining is caused by two toxins (toxin A and toxin B) that are expressed by some C. difficile strains. Toxin A and toxin B are glucosyltransferases that target small host GTPases in the Ras superfamily. They are encoded on the 19.6 kb pathogenicity locus and strains lacking these toxin genes are non-pathogenic. Toxinogenic strains can be further classified into toxinotypes according to sequence variability within the pathogenicity locus. Both toxins contribute to CDI, as shown by using isogenic mutants that produced either toxin A or toxin B alone and were cytotoxic in vitro and virulent in vivo (Kuehne, S. A., et al., Nature, 2010. 467(7316): p. 711-3). A vaccine prototype based on inactivated toxins A and B (toxoids) and anti-toxin monoclonal antibodies are being studied for their effectiveness in preventing recurrent CDI.

Toxin A and toxin B are structurally related, large toxins of MW˜300 kDa, and consist of an amino-terminal catalytic domain (glucosyltransferase), a central peptidase C80 domain, a translocation domain, and multiple carboxy-terminal β-hairpin repeats. The mechanism of action of the clostridial toxins has been shown to involve binding of these β-hairpin repeats to carbohydrates present on the surface of gastrointestinal cells, endopeptidase-mediated cleavage, and internalization of the catalytic domain (Pfeifer, G., et al., J. Biol. Chem., 2003. 278(45): p. 44535-41).

Some C. difficile strains produce a binary toxin which possesses ADP-ribosyltransferase activity. Although its role in pathogenesis is unclear, the presence of binary toxin is a good marker for the epidemic outbreak strain BI/NAP1/027. The binary toxin consists of two subunits, which are the actin ADP-ribosyltransferase binary toxin A chain and the pore-forming binary toxin B chain. They are secreted from the bacterial cells as separate polypeptides and have the potential to combine to form a potent cytotoxin which has been shown to kill Vero cells (Sundriyal, A., et al., Protein Expr. Purif., 2010. 74(1): p. 42-8).

Rapid and accurate CDI diagnosis is important for patient care, infection control and surveillance. The C. difficile toxins A and B are of high clinical diagnostic relevance since they are sufficiently pathogen-specific targets and the demonstration of their presence is important for CDI diagnosis. All currently used CDI diagnostic tests are qualitative and belong to one of three types, (i) cytotoxin assay (tissue culture), (ii) non-molecular toxin tests (EIA), and (iii) molecular tests (PCR).

The tissue culture-based cytotoxin assay is considered the gold standard, but is cumbersome and not routinely performed by most clinical laboratories. In essence, this assay detects C. difficile toxin via the toxin's cytopathic effect in cell culture that can be neutralized with specific anti-sera. The cytotoxicity assay detects as little as 10 pg of toxin B and is the recommended confirmatory test for 510(k) submissions in the “Draft Guidance for Industry and Food and Drug Administration Staff Establishing the Performance Characteristics of In Vitro Diagnostic Devices for the Detection of Clostridium difficile” that was released in November 2010 FDA, http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/uc m234868.htm.2010.

Molecular tests for CDI are available from several diagnostic companies. The Cepheid GeneXpert™ test is based on multiplex PCR (tcdB, cdt, tcdC), with advertised sensitivity and specificity of >95% and time-to-result of 30 min. The Meridian Illumigene™ C. difficile test detects the presence of the toxin producing region by isothermal loop amplification and advertised to provide results in under an hour. The BD GeneOhm™ C diff assay is a real-time PCR method for the detection of toxin B gene (tcdB) direct from stool samples, with an assay protocol time of less than two hours, sensitivity of 93.8% and specificity of 95.5%. Gen-Probe offers the Prodesse ProGastro Cd test which also detects the toxin B gene (tcdB) by PCR and is advertised to provide results in three hours with a sensitivity of 91.7% and specificity of 94.7%.

Non-molecular tests for C. difficile toxin detection in stool samples from patients with suspected CDI are also available. Enzyme immunoassays (EIAs) are the most widely used rapid detection methods for C. difficile common antigen and toxin A/B antigens, but traditional EIAs have modest sensitivity and specificity. Among the well-type EIAs, the Meridian Premier™ Toxins A/B test and the Techlab TOX A/B II™ test are considered the best-performing ELISAs and detect both toxins in stool specimens in less than 1 hour. These assays had about 80% sensitivity and 98% specificity when tested independently. The toxin B antibodies for the Premier™ Toxins A/B (Meridian) and for the C. difficile TOX A/B II™ (TechLab) were able to detect 125 pg and 250 pg of toxin B, respectively, when tested side by side (Novak-Weekley, S. M. and M. H. Hollingsworth. Clin Vaccine Immunol, 2008. 15(3): p. 575-8). Many other well-type EIAs assays have been brought to market (GA's C. difficile antigen, R-Biopharm's Ridascreen™ Toxin A/B; Remel's ProSpect™ Toxin A/B) but are used less often in the U.S. Membrane EIA assays performed with lateral flow devices are the Meridian ImmunoCard™ Toxins A&B, the Techlab Tox A/B Quik Chek™, and the Remel Xpect™ assays.

There is one automated test on the market, bioMérieux's VIDAS™ C. difficile Toxin A&B, which combines toxin testing and culture based identification with the API® 20A strip and automated bacterial genotyping with the DiversiLab® system.

Aptamer-based C. difficile toxin tests, like EIAs, have the advantage over molecular tests that they do not require big investments in equipment or expensive reagents. Aptamers have several distinct advantages over antibodies that are currently used in non-molecular assays, such as EIAs: aptamers generally have lower molecular weight, provide higher multiplexing capabilities (low cross-reactivity, universally-applicable assay conditions), chemical stability (to heat, drying, and solvents, reversible renaturation), provide ease of reagent manufacturing, consistent lot-to-lot performance and can be produced at lower cost.

Aptamers can be generated against virtually any protein target, not only toxins A/B, but also binary toxin for which there is no antibody-based test of which Applicants are aware. Detection and read-out methods can be the same as for existing tests, thus minimizing equipment needs and training requirements.

SUMMARY

The present disclosure provides various aptamers that bind to toxins produced by C. difficile. Included are diagnostic kits and diagnostic methods comprising such aptamers; and methods of making and using such aptamers.

The provided aptamers bind to C. difficile toxin A, toxin B, binary toxin A chain, or binary toxin B chain. Diagnostic methods are provided for detecting a C. difficile toxin which comprise an aptamer to a toxin produced by C. difficile including but are not limited to pull-down assays, dot blot assays, PCR assays and sandwich assays.

The provided aptamers optionally comprise at least one pyrimidine modified at a C-5 position and may comprise at least one addition chemical modification. Also provided are aptamers and methods for identifying or producing such aptamers which bind to a C. difficile toxin which have a slow off-rate from the toxin. Further provided are aptamers and methods for identifying or producing such aptamers which bind to a C. difficile toxin which have nuclease resistance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates the crystal structure of C. difficile toxin A (rTcdA) including five carboxy-terminal receptor-binding repeats (Ho, J. G., et al. Proc. Natl. Acad. Sci. USA, 2005. 102(51): p. 18373-8); shows the purification of toxin A as a recombinant tagged protein purified via affinity chromatography with Ni-NTA agarose and Strep•Tactin resin using the His-tag and Strep tag of toxin A; and PCR amplification of corresponding portion of the toxin gene encoding toxin A (tcdA). FIG. 1B illustrates the crystal structure of C. difficile toxin B (rTcdB) amino-terminal catalytic domain (Reinert, D. J. et al. (2005), J. Mol. Biol. 351: 973-981); shows the purification of toxin B as a recombinant tagged protein purified via affinity chromatography with Ni-NTA agarose and Strep•Tactin resin using the His-tag and Strep tag of toxin B; and PCR amplification of corresponding portion of the toxin gene encoding toxin B (tcdB). FIG. 1C illustrates the crystal structure of the full-length C. difficile binary toxin A chain (rCdtA) (Sundriyal, A., et al., J. Biol. Chem. 2009. 284(42): p. 28713-9); shows the purification of binary toxin A chain as a recombinant tagged protein purified via affinity chromatography with Ni-NTA agarose and Strep•Tactin resin using the His-tag and Strep tag of the binary toxin A chain; and PCR amplification of corresponding portion of the toxin gene encoding the binary toxin A chain (cdtA). FIG. 1D illustrates the modeled structure of the full-length C. difficile binary toxin B chain (rCdtB); shows the purification of binary toxin B chain as a recombinant tagged protein purified via affinity chromatography with Ni-NTA agarose and Strep•Tactin resin using the His-tag and Strep tag of the binary toxin B chain; and PCR amplification of corresponding portion of the toxin gene encoding the binary toxin B chain (cdtB).

FIG. 2A illustrates the results of a pull-down assay of recombinant and native toxin A using toxin A aptamers showing high specificity over the control proteins toxin B or BSA. FIG. 2B illustrates the results of a pull-down assay of recombinant and native toxin B using toxin B aptamers showing high specificity over the control proteins recombinant and native toxin A. FIG. 2C illustrates the results of a pull-down assay of binary toxin with aptamers to binary toxin A chain showing specificity for binary toxin A chain over binary toxin B chain and control protein BSA. FIG. 2D illustrates the results of a pull-down assay of recombinant and native toxin A as well as recombinant and native toxin B with random aptamers showing no proteins present in the pull-down fraction.

FIG. 3A illustrates detection of C. difficile toxin A on dot blots using biotinylated aptamers to toxin A and streptavidin-alkaline phosphatase conjugate. FIG. 3B illustrates detection of C. difficile toxin B on dot blots using biotinylated aptamers to toxin B and streptavidin-alkaline phosphatase conjugate.

FIG. 4A illustrates quantitative detection of toxin A by qPCR of aptamers eluted from a sample containing aptamers in complex with toxin A wherein unbound aptamers have been removed before quantitative measurement of aptamers as proxy measurement for toxin A. FIG. 4B illustrates quantitative detection of toxin B by qPCR of aptamers eluted from a sample containing aptamers in complex with toxin B wherein unbound aptamers have been removed before quantitative measurement of aptamers as proxy measurement for toxin B.

FIG. 5A illustrates results of detection of C. difficile toxin A via streptavidin plate sandwich (aptamer-target-antibody) assay, using biotinylated aptamers to toxin A and mouse monoclonal antibodies to toxin A detected with goat-anti-mouse antibodies. FIG. 5B illustrates results of detection of C. difficile toxin B via streptavidin plate sandwich (aptamer-target-antibody) assay, using biotinylated aptamers to toxin B and mouse monoclonal antibodies to toxin B detected with goat-anti-mouse antibodies.

FIG. 6 illustrates results of detection of C. difficile toxin A and B via sandwich (antibody-target-aptamer) assays on nitrocellulose, wherein monoclonal antibodies are spotted onto nitrocellulose and air dried, blocked, samples containing toxin A or B added, washed, biotinylated aptamers added, washed, and developed with streptavidin-alkaline phosphatase conjugate.

FIG. 7 illustrates results of detection of C. difficile binary toxin A chain in a sandwich (aptamer-target-aptamer) assay with a first biotinylated aptamer to binary toxin A chain attached a streptavidin bead, addition of target, and addition of a second, radiolabeled aptamer to binary toxin A chain.

FIG. 8 illustrates relevant steps of a Catch 1-Catch 2 assay.

FIG. 9 illustrates examples of C-5 modified pyrimidines which may be used in the methods of making aptamers described herein.

DETAILED DESCRIPTION

Reference will now be made in detail to representative embodiments of the invention. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that the invention is not intended to be limited to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in and are within the scope of the practice of the present invention. The present invention is in no way limited to the methods and materials described.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art(s) to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.

All publications, published patent documents, and patent applications cited in this disclosure are indicative of the level of skill in the art(s) to which the disclosure pertains. All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.

As used in this disclosure, including the appended claims, the singular forms “a,” “an,” and “the” include plural references, unless the content clearly dictates otherwise, and are used interchangeably with “at least one” and “one or more.” Thus, reference to “an aptamer” includes mixtures of aptamers, and the like.

As used herein, the term “about” represents an insignificant modification or variation of the numerical value such that the basic function of the item to which the numerical value relates is unchanged.

As used herein, the term “aptamer clone” refers to an aptamer of a particular nucleotide sequence. Aptamer clones are identified herein by “Aptamer ID No.” as well as by “SEQ ID NO:.”

As used herein, “competitor molecule” and “competitor” are used interchangeably to refer to any molecule that can form a non-specific complex with a non-target molecule. A “competitor molecule” or “competitor” is a set of copies of one type or species of molecule. “Competitor molecules” or “competitors” refer to more than one such set of molecules. Competitor molecules include oligonucleotides, polyanions (e.g., heparin, single-stranded salmon sperm DNA, and polydextrans (e.g., dextran sulphate)), abasic phosphodiester polymers, dNTPs, and pyrophosphate. In the case of a kinetic challenge that uses a competitor, the competitor can also be any molecule that can form a non-specific complex with an aptamer. Such competitor molecules include polycations (e.g., spermine, spermidine, polylysine, and polyarginine) and amino acids (e.g., arginine and lysine).

As used in tables 4, 6, 8 and 10, the term “count” refers to the number of occurrences of a particular aptamer sequence among all aptamers that were cloned and sequenced from a pool that resulted from SELEX.

As used herein, the term “dot blot” refers to an assay wherein a mixture containing the target molecule to be detected is applied directly onto a substrate as a dot followed by detection of the presence of the target molecule by an affinity molecule, wherein the affinity molecule may be, but is not limited to, an aptamer or antibody.

The term “each” when used herein to refer to a plurality of items is intended to refer to at least two of the items. It need not require that all of the items forming the plurality satisfy an associated additional limitation.

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “contains,” “containing,” and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, product-by-process, or composition of matter that comprises, includes, or contains an element or list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, product-by-process, or composition of matter.

As used herein, “consensus sequence,” when used in reference to a series of related nucleic acids, refers to a nucleotide sequence that reflects the most common choice of base at each position in the sequence where the series of related nucleic acids has been subjected to mathematical and/or sequence analysis.

As used herein, the term “nucleotide” refers to a ribonucleotide or a deoxyribonucleotide, or a modified form thereof, as well as an analog thereof. Nucleotides include species that include purines (e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs) as well as pyrimidines (e.g., cytosine, uracil, thymine, and their derivatives and analogs).

As used herein, “nucleic acid,” “oligonucleotide,” and “polynucleotide” are used interchangeably to refer to a polymer of nucleotides and include DNA, RNA, DNA/RNA hybrids and modifications of these kinds of nucleic acids, oligonucleotides and polynucleotides, wherein the attachment of various entities or moieties to the nucleotide units at any position are included. The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” include double- or single-stranded molecules as well as triple-helical molecules. Nucleic acid, oligonucleotide, and polynucleotide are broader terms than the term aptamer and, thus, the terms nucleic acid, oligonucleotide, and polynucleotide include polymers of nucleotides that are aptamers but the terms nucleic acid, oligonucleotide, and polynucleotide are not limited to aptamers.

As used herein, the terms “modify”, “modified”, “modification”, and any variations thereof, when used in reference to an oligonucleotide, means that at least one of the four constituent nucleotide bases (i.e., A, G, T/U, and C) of the oligonucleotide is an analog or ester of a naturally occurring nucleotide. In some embodiments, the modified nucleotide confers nuclease resistance to the oligonucleotide. A pyrimidine with a substitution at the C-5 position is an example of a modified nucleotide. Modifications can include backbone modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine, and the like. Modifications can also include 3′ and 5′ modifications, such as capping. Other modifications can include substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, and those with modified linkages (e.g., alpha anomeric nucleic acids, etc.). Further, any of the hydroxyl groups ordinarily present on the sugar of a nucleotide may be replaced by a phosphonate group or a phosphate group; protected by standard protecting groups; or activated to prepare additional linkages to additional nucleotides or to a solid support. The 5′ and 3′ terminal OH groups can be phosphorylated or substituted with amines, organic capping group moieties of from about 1 to about 20 carbon atoms, polyethylene glycol (PEG) polymers in one embodiment ranging from about 10 to about 80 kDa, PEG polymers in another embodiment ranging from about 20 to about 60 kDa, or other hydrophilic or hydrophobic biological or synthetic polymers. In one embodiment, modifications are of the C-5 position of pyrimidines. These modifications can be produced through an amide linkage directly at the C-5 position or by other types of linkages.

Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. As noted above, one or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R′, P(O)OR′, CO or CH₂ (“formacetal”), in which each R or R′ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. Substitution of analogous forms of sugars, purines, and pyrimidines can be advantageous in designing a final product, as can alternative backbone structures like a polyamide backbone, for example.

As used herein, the term “C-5 modified pyrimidine” refers to a pyrimidine with a modification at the C-5 position including, but not limited to, those moieties illustrated in FIG. 9. Examples of a C-5 modified pyrimidine include those described in U.S. Pat. Nos. 5,719,273 and 5,945,527. Examples of a C-5 modification include substitution of deoxyuridine at the C-5 position with a substituent independently selected from: benzylcarboxyamide (alternatively benzylaminocarbonyl) (Bn), naphthylmethylcarboxyamide (alternatively naphthylmethylaminocarbonyl) (Nap), tryptaminocarboxyamide (alternatively tryptaminocarbonyl) (Trp), tyrosylcarboxyamide (alternatively tyrosylaminocarbonyl) (Tyr), 2-naphthylmethylcarboxyamide (alternatively 2-naphthylmethylaminocarbonyl) (2Nap) and phenethyl-1-carboxyamide (alternatively phenethyl-1-aminocarbonyl) (PE), as illustrated immediately below.

Chemical modifications of a C-5 modified pyrimidine can also be combined with, singly or in any combination, 2′-position sugar modifications, modifications at exocyclic amines, and substitution of 4-thiouridine and the like.

Representative C-5 modified pyrimidines include: 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU), 5-(N-benzylcarboxyamide)-2′-O-methyluridine, 5-(N-benzylcarboxyamide)-2′-fluorouridine, 5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU), 5-(N-tryptaminocarboxyamide)-2′-O-methyluridine, 5-(N-tryptaminocarboxyamide)-2′-fluorouridine, 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU), 5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine, 5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine, 5-(N-tyrosylcarboxyamide)-2′-deoxyuridine (TyrdU), 5-(N-tyrosylcarboxyamide)-2′-O-methyluridine, 5-(N-tyrosylcarboxyamide)-2′-fluorouridine, 5-(N-(2-naphthylmethyl)carboxyamide)-2′-deoxyuridine (2NapdU), 5-(N-(2-naphthylmethyl)carboxyamide)-2′-O-methyluridine, 5-(N-(2-naphthylmethyl)carboxyamide)-2′-fluorouridine, 5-(N-phenethyl-1-carboxyamide)-2′-deoxyuridine (PEdU), 5-(N-phenethyl-1-carboxyamide)-2′-O-methyluridine, or 5-(N-phenethyl-1-carboxyamide)-2′-fluorouridine.

If present, a modification to the nucleotide structure can be imparted before or after assembly of a polymer. A sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.

As used herein, the term “at least one pyrimidine,” when referring to modifications of a nucleic acid, refers to one or more, two or more, three or more, four or more, five or more, or all pyrimidines in the nucleic acid, indicating that any or all occurrences of any or all of C, T, or U in a nucleic acid may be modified or not.

As used herein, the terms “kinetically challenge” and “kinetic challenge” refer to a process of enrichment for an aptamer affinity complex from a set of complexes that includes an aptamer affinity complex and non-specific complexes, by applying kinetic pressure and making use of the different affinity characteristics of the constituents of such classes of complexes, including dissociation rates. A kinetic challenge generally results in an increase in specificity, since aptamer-non-target complexes are typically reduced compared to aptamer-target complexes. As used herein, the term “kinetic pressure” refers to a means for providing an opportunity for the natural dissociation of complexes and/or inhibiting the rebinding of molecules that dissociate from a complex naturally. Kinetic pressure can be applied by the addition of a competitor molecule, or by sample dilution, or by extensive washes when complexes are bound to a solid support, or by any other means known to one skilled in the art. As one of ordinary skill in the art will appreciate, because a kinetic challenge generally depends upon the differing dissociation rates of aptamer affinity complexes and aptamer-non-target complexes, the duration of the kinetic challenge is chosen so as to retain a high proportion of aptamer affinity complexes while substantially reducing the number of aptamer-non-target complexes. For a kinetic challenge to be effective, the dissociation rate for the aptamer affinity complex is preferably significantly lower than those for aptamer-non-target complexes. Since an aptamer can be selected to include particular properties, the constituents of an aptamer affinity complex can be designed to have a comparatively low dissociation rate, i.e. slow off rate.

As used herein, “nucleic acid ligand,” “aptamer,” and “clone” are used interchangeably to refer to a non-naturally occurring nucleic acid that has a desirable action on a target molecule. A desirable action includes, but is not limited to, binding of the target, catalytically changing the target, reacting with the target in a way that modifies or alters the target or the functional activity of the target, covalently attaching to the target (as in a suicide inhibitor), and facilitating the reaction between the target and another molecule. In one embodiment, the action is specific binding affinity for a target molecule, such target molecule being a three dimensional chemical structure other than a polynucleotide that binds to the nucleic acid ligand through a mechanism which is independent of Watson/Crick base pairing or triple helix formation, wherein the aptamer is not a nucleic acid having the known physiological function of being bound by the target molecule. Aptamers to a given target include nucleic acids that are identified from a candidate mixture of nucleic acids, where the aptamer is a ligand of the target, by a method comprising: (a) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to other nucleic acids in the candidate mixture can be partitioned from the remainder of the candidate mixture; (b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and (c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby aptamers of the target molecule are identified. It is recognized that affinity interactions are a matter of degree; however, in this context, the “specific binding affinity” of an aptamer for its target means that the aptamer binds to its target generally with a much higher degree of affinity than it binds to other, non-target, components in a mixture or sample. An “aptamer” or “nucleic acid ligand” is a set of copies of one type or species of nucleic acid molecule that has a particular nucleotide sequence. An aptamer can include any suitable number of nucleotides. “Aptamers” refer to more than one such set of molecules. Different aptamers can have either the same or different numbers of nucleotides. Aptamers may be DNA or RNA and may be single stranded, double stranded, or contain double stranded or triple stranded regions.

As used herein, IUPAC nucleotide ambiguity codes are: M=A or C; R=A or G; W=A or N; S=C or G; Y=C or N; K=G or N(N represents the pool-specific modified dU).

As used herein, “plateau” refers to a region of a binding curve (in which the fraction of aptamers bound increases up the y-axis and the concentration of target increases to the right on the x-axis) where a plateau is reached as increasing target concentration causes relatively little change in the fraction of aptamers bound to target. The plateau percentage provided herein is relative to 100% of aptamers being bound to target.

As used herein, “protein” is used synonymously with “peptide”, “polypeptide”, or “peptide fragment.” A “purified” polypeptide, protein, peptide, or peptide fragment is substantially free of cellular material or other contaminating proteins from the cell, tissue, or cell-free source from which the amino acid sequence is obtained, or substantially free from chemical precursors or other chemicals when chemically synthesized.

As used herein, “pull-down assay” refers to an assay which comprises removal of a target from solution wherein the removal is accomplished by a selective affinity interaction between the target and a second molecule. In one embodiment, the molecule having selective affinity for the target molecule is an aptamer. In another embodiment, the molecule having selective affinity for the target is an antibody.

As used herein, “PCR” refers to a polymerase chain reaction used to amplify the copy number of a DNA molecule. As used herein, “qPCR” or “quantitative PCR” refers to a polymerase chain reaction that is used to amplify and simultaneously quantify a targeted DNA molecule.

As used herein, “sandwich assay” refers to an assay capable of detecting the presence of or quantitating the amount of a target of interest. The assay requires the use of two different affinity molecules capable of binding two different, non-overlapping (noncompetitive) regions on a target of interest. Affinity molecules include but are not limited to aptamers and antibodies.

As used herein, “substrate” refers to a surface, including but not limited to the surface of a plate, bead or membrane to which an organic molecule can attach. A substrate may or may not comprise a first molecule that mediates attachment of a second molecule, such as a substrate comprising streptavidin which can mediate attachment of biotin or a molecule comprising a biotin moiety. In one embodiment, the substrate is nitrocellulose.

As used herein, “test sample” refers to a sample in which the presence or amount of one or more analytes of interest (e.g. C. difficile toxin A, toxin B, binary toxin A chain, or binary toxin B chain) are unknown and to be determined in an assay, preferably a diagnostic test comprising an aptamer. In one embodiment, the test sample can be a “biological sample” such as cellular and non-cellular biological material, including, but not limited to, tissue samples, blood, serum, other bodily fluids, and excrement. In another embodiment, the test sample can be an “environmental sample” which can be obtained from water, soil or air. Normally no prior culturing is necessary for detection of C. difficile in environmental samples.

The SELEX Method

The terms “SELEX” and “SELEX process” are used interchangeably herein to refer generally to a combination of (1) the selection of nucleic acids that interact with a target molecule in a desirable manner, for example binding with high affinity to a protein, with (2) the amplification of those selected nucleic acids. The SELEX process can be used to identify aptamers with high affinity to a specific target molecule or biomarker.

SELEX generally includes preparing a candidate mixture of nucleic acids, binding of the candidate mixture to the desired target molecule to form an affinity complex, separating the affinity complexes from the unbound candidate nucleic acids, separating and isolating the nucleic acid from the affinity complex, purifying the nucleic acid, and identifying a specific aptamer sequence. The process may include multiple rounds to further refine the affinity of the selected aptamer. The process can include amplification steps at one or more points in the process. See, e.g., U.S. Pat. No. 5,475,096, entitled “Nucleic Acid Ligands.” The SELEX process can be used to generate an aptamer that covalently binds its target as well as an aptamer that non-covalently binds its target. See, e.g., U.S. Pat. No. 5,705,337 entitled “Systematic Evolution of Nucleic Acid Ligands by Exponential Enrichment: Chemi-SELEX.”

The SELEX process can be used to identify high-affinity aptamers containing modified nucleotides that confer improved characteristics on the aptamer, such as, for example, improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified aptamers containing modified nucleotides are described in U.S. Pat. No. 5,660,985, entitled “High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,” which describes oligonucleotides containing nucleotide derivatives chemically modified at the 5′- and 2′-positions of pyrimidines. U.S. Pat. No. 5,580,737, see supra, describes highly specific aptamers containing one or more nucleotides modified with 2′-amino (2′-NH₂), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). See also, U.S. Patent Application Publication No. 2009/0098549, entitled “SELEX and PHOTOSELEX,” which describes nucleic acid libraries having expanded physical and chemical properties and their use in SELEX and photoSELEX.

The nuclease resistant oligonucleotides include at least one pyrimidine modified at the C-5 position with a group selected from those set forth in FIG. 9. In various embodiments, the modifications include substitution of deoxyuridine at the C-5 position with a substituent independently selected from: benzylcarboxyamide (Bn), naphthylmethylcarboxyamide (Nap), tryptaminocarboxyamide (Trp), tyrosylcarboxyamide (Tyr), (2-naphthylmethyl)carboxyamide (2Nap), and phenethyl-1-carboxyamide (PE) as illustrated above.

SELEX can also be used to identify aptamers that have desirable off-rate characteristics. See U.S. Patent Publication No. 2009/0004667, entitled “Method for Generating Aptamers with Improved Off-Rates,” which describes improved SELEX methods for generating aptamers that can bind to target molecules. Methods for producing aptamers and photoaptamers having slower rates of dissociation from their respective target molecules are described. The methods involve contacting the candidate mixture with the target molecule, allowing the formation of nucleic acid-target complexes to occur, and performing a slow off-rate enrichment process wherein nucleic acid-target complexes with fast dissociation rates dissociate and do not reform, while complexes with slow dissociation rates remain intact. Additionally, the methods include the use of modified nucleotides in the production of candidate nucleic acid mixtures to generate aptamers with improved off-rate performance (see U.S. Patent Publication No. 2009/0098549, entitled “SELEX and PhotoSELEX”).

“Target” or “target molecule” or “target” refers herein to any compound upon which a nucleic acid can act in a desirable manner. A target molecule can be a protein, peptide, nucleic acid, carbohydrate, lipid, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, pathogen, toxic substance, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient, growth factor, cell, tissue, any portion or fragment of any of the foregoing, etc., without limitation. Virtually any chemical or biological effector may be a suitable target. Molecules of any size can serve as targets. A target can also be modified in certain ways to enhance the likelihood or strength of an interaction between the target and the nucleic acid. A target can also include any minor variation of a particular compound or molecule, such as, in the case of a protein, for example, minor variations in amino acid sequence, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component, which does not substantially alter the identity of the molecule. A “target molecule” or “target” is a set of copies of one type or species of molecule or multimolecular structure that is capable of binding to an aptamer. “Target molecules” or “targets” refer to more than one such set of molecules. Embodiments of the SELEX process in which the target is a peptide are described in U.S. Pat. No. 6,376,190, entitled “Modified SELEX Processes Without Purified Protein.” In the instant case, the targets include C. difficile toxin A, toxin B, binary toxin, binary toxin A chain, or binary toxin B chain.

Methods of Identifying or Producing an Aptamer to a C. difficile Toxin

The present disclosure provides methods of identifying or producing a slow off-rate aptamer that binds to a toxin produced by C. difficile wherein said toxin is selected from toxin A, toxin B, binary toxin A chain, and binary toxin B chain, the method comprising:

(a) preparing a candidate mixture of nucleic acids, wherein the candidate mixture comprises modified nucleic acids in which one, several or all pyrimidines in at least one, or each, nucleic acid of the candidate mixture comprises a chemical modification at a C-5 position; (b) contacting the candidate mixture with a target which is said toxin produced by C. difficile and exposing the candidate mixture to a slow off-rate enrichment process, wherein nucleic acids having a slow rate of dissociation from the target relative to other nucleic acids in the candidate mixture bind the target, forming nucleic acid-target molecule complexes; (c) partitioning slow off-rate nucleic acids from the candidate mixture; and (d) amplifying the slow off-rate nucleic acids to yield a mixture of nucleic acids enriched in nucleic acid sequences that are capable of binding to the target molecule with a slow off-rate, whereby a slow off-rate aptamer to the target molecule may be identified. The methods of identifying or producing a slow off rate aptamer to a C. difficile toxin may comprise at least one pyrimidine wherein a chemical modification at a C-5 position is independently chosen from at least one of the modifications shown in FIG. 9. The methods of identifying or producing a slow off rate aptamer to a C. difficile toxin may comprise at least one pyrimidine wherein a chemical modification at a C-5 position is independently chosen from benzylcarboxyamide, naphthylmethylcarboxyamide, tryptaminocarboxyamide, tyrosylcarboxyamide, 2-naphthylmethylcarboxyamide and phenethyl-1-carboxyamide. The methods of identifying or producing a slow off rate aptamer to a C. difficile toxin may comprise at least one additional chemical modification, wherein said at least one additional chemical modification is a chemical substitution at one or more positions independently selected from the group consisting of a ribose position, a deoxyribose position, a phosphate position, and a base position. Further, the methods of identifying or producing a slow off rate aptamer to a C. difficile toxin may comprise at least one additional chemical modification, wherein said at least one additional chemical modification is independently selected from the group consisting of a 2′-position sugar modification, a 2′-amino (2′—NH₂), a 2′-fluoro (2′-F), a 2′-β-methyl (2′-OMe) a modification at a cytosine exocyclic amine, a substitution of 5-bromouracil, a substitution of 5-bromodeoxyuridine, a substitution of 5-bromodeoxycytidine, a backbone modification, methylation, a 3′ cap, and a 5′ cap. The methods of identifying or producing a slow off rate aptamer to a C. difficile toxin may comprise a slow off-rate enrichment process selected from incubation of a candidate mixture with a competitor molecule, dilution of a candidate mixture, or dilution of a candidate mixture in the presence of a competitor molecule.

The present disclosure further provides methods of producing an aptamer having a slow rate of dissociation from a toxin produced by C. difficile wherein said toxin is selected from toxin A, toxin B, binary toxin A chain, and binary toxin B chain, said method comprising the step of preparing or synthesizing an aptamer based on a nucleic acid sequence identified by a process comprising the steps of: (a) preparing a candidate mixture of nucleic acids, wherein the candidate mixture comprises modified nucleic acids in which one, several or all pyrimidines in at least one, or each, nucleic acid of the candidate mixture comprises a chemical modification at a C-5 position; (b) contacting the candidate mixture with a target which is said toxin produced by C. difficile and exposing the candidate mixture to a slow off-rate enrichment process, wherein nucleic acids having a slow rate of dissociation from the target molecule relative to other nucleic acids in the candidate mixture bind the target molecule, forming nucleic acid-target molecule complexes; (c) partitioning slow off-rate nucleic acids from the candidate mixture; and (d) amplifying the slow off-rate nucleic acids to yield a mixture of nucleic acids enriched in nucleic acid sequences that are capable of binding to the target molecule with a slow off-rate, whereby a slow off-rate aptamer to the target molecule is identified. Such methods of producing an aptamer having a slow rate of dissociation from a C. difficile toxin may comprise at least one pyrimidine wherein a chemical modification at a C-5 position is independently chosen from at least one of the modifications shown in FIG. 9. The methods of producing an aptamer having a slow rate of dissociation from a C. difficile toxin may comprise at least one pyrimidine wherein a chemical modification at a C-5 position is independently chosen from benzylcarboxyamide, naphthylmethylcarboxyamide, tryptaminocarboxyamide, tyrosylcarboxyamide, 2-naphthylmethylcarboxyamide and phenethyl-1-carboxyamide. The methods of producing an aptamer having a slow rate of dissociation from a C. difficile toxin may comprise at least one additional chemical modification, wherein said at least one additional chemical modification is a chemical substitution at one or more positions independently selected from the group consisting of a ribose position, a deoxyribose position, a phosphate position, and a base position. Further, the methods of producing an aptamer having a slow rate of dissociation from a C. difficile toxin may comprise at least one additional chemical modification, wherein said at least one additional chemical modification is independently selected from the group consisting of a 2′-position sugar modification, a 2′-amino (2′-NH₂), a 2′-fluoro (2′-F), a 2′-β-methyl (2′-OMe) a modification at a cytosine exocyclic amine, a substitution of 5-bromouracil, a substitution of 5-bromodeoxyuridine, a substitution of 5-bromodeoxycytidine, a backbone modification, methylation, a 3′ cap, and a 5′ cap. The methods of producing an aptamer having a slow rate of dissociation from a C. difficile toxin may comprise a slow off-rate enrichment process selected from incubation of a candidate mixture with a competitor molecule, dilution of a candidate mixture, or dilution of a candidate mixture in the presence of a competitor molecule.

The present disclosure further provides methods for producing a nuclease resistant aptamer that binds to a toxin produced by C. difficile wherein said toxin is selected from toxin A, toxin B, binary toxin A chain, and binary toxin B chain, the method comprising preparing or synthesizing said nuclease resistant aptamer based on a nucleic acid sequence identified by a process comprising: (a) preparing a candidate mixture of modified nucleic acids, wherein the candidate mixture comprises modified nucleic acids in which at least one pyrimidine in at least one, or in each, nucleic acid of the candidate mixture comprises a chemical modification at a C-5 position; (b) contacting the candidate mixture with a target which is said toxin produced by C. difficile, wherein nucleic acids having an increased affinity to the target molecule relative to other nucleic acids in the candidate mixture bind the target molecule, forming nucleic acid-target molecule complexes; (c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and (d) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched in nucleic acid sequences that are capable of binding to the target molecule with increased affinity and that are nuclease resistant, whereby a nuclease resistant aptamer to the target molecule is identified. The methods of producing a nuclease resistant aptamer to a C. difficile toxin may comprise at least one pyrimidine wherein a chemical modification at a C-5 position is independently chosen from at least one of the modifications shown in FIG. 9. The methods of producing a nuclease resistant aptamer to a C. difficile toxin may comprise at least one pyrimidine wherein a chemical modification at a C-5 position is independently chosen from benzylcarboxyamide, naphthylmethylcarboxyamide, tryptaminocarboxyamide, tyrosylcarboxyamide, 2-naphthylmethylcarboxyamide and phenethyl-1-carboxyamide. The methods of producing a nuclease resistant aptamer to a C. difficile toxin may comprise at least one additional chemical modification, wherein said at least one additional chemical modification is a chemical substitution at one or more positions independently selected from the group consisting of a ribose position, a deoxyribose position, a phosphate position, and a base position. Further, the methods of producing a nuclease resistant aptamer to a C. difficile toxin may comprise at least one additional chemical modification, wherein said at least one additional chemical modification is independently selected from the group consisting of a 2′-position sugar modification, a 2′-amino (2′-NH₂), a 2′-fluoro (2′-F), a 2′-O-methyl (2′-OMe) a modification at a cytosine exocyclic amine, a substitution of 5-bromouracil, a substitution of 5-bromodeoxyuridine, a substitution of 5-bromodeoxycytidine, a backbone modification, methylation, a 3′ cap, and a 5′ cap.

Aptamers

The aptamers to C. difficile toxins of the instant disclosure were identified using the improved SELEX method for identifying aptamers having slow off-rates, as described above. The form of C. difficile toxins used in the selection process were recombinant toxins prepared by PCR amplification of desired gene fragments from C. difficile genomic DNA, as described in Example 1.

SELEX was performed using purified His-tag proteins obtained from over-expression of cloned C. difficile toxin gene fragments. Libraries of 40mer random sequences were used that contained one of six modified nucleotides, 5-tyrosylcarboxyamide-dU (TyrdU), 5-benzylcarboxyamide-dU (BndU), 5-naphthylmethylcarboxyamide-dU (NapdU), 5-tryptaminocarboxyamide-dU (TrpdU), 5-(2-naphthylmethyl)carboxyamide (2NapdU), or 5-phenethyl-1-carboxyamide (PEdU) instead of dU. Seven or eight rounds of selection were carried out, and a kinetic challenge with dextran sulfate was applied in rounds 2-8. The aptamer pools obtained after the last round of SELEX were tested for affinity to their targets in filter binding assays, and the K_(d)'s and plateaus were determined (Table 2). All pools with sufficient affinity (K_(d) of ˜10 nM or below) were cloned and the sequences of at least 48 clones per pool were determined.

Aptamers to Toxin A

For toxin A, the aptamer pool 4943 (TrpdU) had excellent affinity with a K_(d)=2.42 nM. Pools 4936 (TyrdU) and 4939 (NapdU) were active, with K_(d)'s of 11.5 and 10.8 nM, respectively. Pools 5564 (2NapdU) and 5577 (2NapdU) had good affinity, with K_(d)'s of 4.63 and 6.40 nM. For toxin A, the aptamer pool 5570 (PEdU) was the best, with a K_(d)=1.61 nM. Aptamer clones with good affinities for toxin A were isolated from all of the pools with TrpdU, TyrdU, NapdU, 2NapdU, and PEdU modified nucleotides (Table 3); the sequences of C. difficile toxin A aptamers are listed on Table 4. In addition to identifying aptamer clones with good binding affinity to toxin A, consensus sequences between such aptamer clones were identified.

The leading aptamer clone from pool 5570 (PEdU) was 5570-54, with K_(d)=0.12 nM for the recombinant toxin A and K_(d)=6.91 nM for native toxin A.

Some aptamer clones demonstrated excellent affinity for both recombinant toxin A and native toxin A, e.g. aptamer clone 4943-51 (TrpdU) had K_(d)=1.23 nM for recombinant toxin A and K_(d)=1.78 nM for native toxin A; aptamer clone 5564-49 (2NapdU) had K_(d)=1.13 nM for recombinant toxin A and K_(d)=1.78 nM for native toxin A.

Some aptamer clones demonstrated relatively little drop off of affinity between recombinant toxin A and native toxin A, e.g. aptamer clone 5577-1 (2NapdU) had K_(d)=1.59 nM for recombinant toxin A and K_(d)=4.97 nM for native toxin A; aptamer clone 5577-3 (2NapdU) had K_(d)=1.73 nM for recombinant toxin A and K_(d)=5.52 nM for native toxin A; aptamer clone 4943-60 (TrpdU) had K_(d)=2.65 nM for recombinant toxin A and K_(d)=4.57 nM for native toxin A.

In addition to identifying aptamer clones with good binding affinity to toxin A, consensus sequences between such aptamer clones were identified.

Aptamers to Toxin B

The affinities of aptamers for toxin B were generally very good and correlated well between the 68.8 kDa amino-terminal catalytic domain of C. difficile toxin B fragment that had been used in SELEX and the 270 kDa native, full-length toxin B. Aptamer clones with sub-nanomolar K_(d)'s for toxin B were isolated from all of the pools with TyrdU, BndU, NapdU, TrpdU, 2NapdU and PEdU modified nucleotides (Table 5); the sequences of C. difficile toxin B aptamers are listed in Table 6, with the best clones shown in bold.

The highest-affinity aptamers were clones with NapdU or TrpdU modified nucleotides. Five aptamers demonstrated very low K_(d)'s of <0.1 nM: aptamer clone 4940-1 (NapdU) had K_(d)=0.04 nM for recombinant toxin B and K_(d)=0.06 for native toxin B; aptamer clone 4940-23 (NapdU) had K_(d)=0.07 nM for recombinant toxin B and K_(d)=0.09 nM for native toxin B; aptamer clone 4940-27 (NapdU) had K_(d)=0.10 nM for recombinant toxin B and K_(d)=0.09 nM for native toxin B; aptamer clone 4944-5 (TrpdU) had K_(d)=0.08 nM for recombinant toxin B and K_(d)=0.09 nM for native toxin B; and aptamer clone 4944-30 (TrpdU) had K_(d)=0.06 nM for recombinant toxin B and K_(d)=0.08 nM for native toxin B. Aptamer clones with good affinities for toxin B were isolated from all of the pools with TrpdU, TyrdU, NapdU, 2NapdU, BndU and PEdU modified nucleotides (Table 5); the sequences of C. difficile toxin B aptamers are listed on Table 6. In addition to identifying aptamer clones with good binding affinity to toxin B, consensus sequences between such aptamer clones were identified.

Aptamers to Binary Toxin (A chain)

SELEX with the recombinant binary toxin A chain (CdtA) yielded active aptamers with TrpdU, 2NapdU and PEdU modified nucleotides (Table 7). The sequences and common sequence patterns of CdtA aptamers are shown in Table 8. Cloning of pool 4758 (TrpdU) revealed clone 4758-6 which comprised 18% of the sequences in that pool and showed good affinity (K_(d)=0.86 nM) to CdtA binary toxin. Twenty sequences from 2NapdU pools were obtained, most of them with subnanomolar affinity, and several sequence patterns shared between these 2NapdU clones were identified. PEdU pools contained five active aptamers.

Aptamers to Binary Toxin (B chain)

SELEX with recombinant binary toxin B chain (CdtB) yielded active aptamers with 2NapdU modified nucleotides (Table 9). The sequences and common sequence patterns of CdtB aptamers are shown in Table 10. The most active clone was 5556-51 K_(d)=1.68 nM.

The present disclosure provides aptamers to toxins produced by C. difficile identified using the SELEX method and listed in Tables 4, 6, 8 and 10. Aptamers to toxins produced by C. difficile that are substantially homologous to any of the listed aptamers and that have a substantially similar ability to bind the respective toxin produced by C. difficile as that of an aptamer selected from the group of aptamers set forth in Tables 4, 6, 8 and 10 are also encompassed by the present disclosure. Further, aptamers to the respective toxin produced by C. difficile that have substantially the same structural form as the aptamers identified herein and that have a substantially similar ability to bind the respective toxin produced by C. difficile as that of an aptamer selected from the group of aptamers set forth in Tables 4, 6, 8 and 10 are also encompassed by the present disclosure.

In one aspect, the present disclosure provides an aptamer that specifically binds to a toxin produced by C. difficile and includes a primary nucleic acid sequence. In one embodiment, the primary nucleic acid sequence is selected from a sequence disclosed in Table 4, 6, 8, or 10. In other embodiments, the primary nucleic acid sequence is selected such that it is at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical to a primary nucleic acid sequence disclosed in Table 4, 6, 8 or 10.

The terms “sequence identity”, “percent sequence identity”, “percent identity”, “% identical”, “% identity”, and variations thereof, when used in the context of two or more nucleic acid sequences, are used interchangeably to refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. For sequence comparisons, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 1981. 2:482, by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 1970. 48:443, by the search for similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA, 1988. 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Ausubel, F. M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987)).

One example of an algorithm that is suitable for determining percent sequence identity is the algorithm used in the basic local alignment search tool (hereinafter “BLAST”), see, e.g. Altschul et al., J. Mol. Biol., 1990. 215:403-410 and Altschul et al., Nucleic Acids Res., 1997. 15:3389-3402. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (hereinafter “NCBI”). The default parameters used in determining sequence identity using the software available from NCBI, e.g., BLASTN (for nucleotide sequences) are described in McGinnis et al., Nucleic Acids Res., 2004. 32:W20-W25.

As used herein, when describing the percent identity of a nucleic acid, such as an aptamer to a toxin produced by C. difficile, the sequence of which is at least, for example, about 95% identical to a reference nucleotide sequence, it is intended that the nucleic acid sequence is identical to the reference sequence except that the nucleic acid sequence may include up to five point mutations per each 100 nucleotides of the reference nucleic acid sequence. In other words, to obtain a desired nucleic acid sequence, the sequence of which is at least about 95% identical to a reference nucleic acid sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or some number of nucleotides up to 5% of the total number of nucleotides in the reference sequence may be inserted into the reference sequence (referred to herein as an insertion). These mutations of the reference sequence to generate the desired sequence may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. The reference (query) sequence may be any one of the entire nucleotide sequences shown in Table 4, 6, 8 or 10, or any fragment of any of these sequences.

In one aspect, a consensus sequence selected from the group consisting of SEQ ID NOs: 5, 11, 15, 23, 28, 32, 47, 66, 75, 83, 90, 95, 98, 110, 124, 125, 134, 139, 145, 151, 152 or 157 can be modified to comprise at least one insertion, one deletion and/or one transposition. In one embodiment, the consensus sequence selected from the group consisting of SEQ ID NOs: 5, 11, 15, 23, 28, 32, 47, 66, 75, 83, 90, 95, 98, 110, 124, 125, 134, 139, 145, 151, 152 or 157 is modified such that at least one nucleotide is inserted into the consensus sequence. In another embodiment, a consensus sequence selected from the group consisting of SEQ ID NOs: 5, 11, 15, 23, 28, 32, 47, 66, 75, 83, 90, 95, 98, 110, 124, 125, 134, 139, 145, 151, 152 or 157 is modified such that at least one nucleotide is deleted from the consensus sequence. In another embodiment, a consensus sequence selected from the group consisting of SEQ ID NOs: 5, 11, 15, 23, 28, 32, 47, 66, 75, 83, 90, 95, 98, 110, 124, 125, 134, 139, 145, 151, 152 or 157 is modified such that at least one nucleotide is transposed from one location in the consensus sequence to another location in the consensus sequence. It is also recognized that a consensus sequence selected from the group consisting of SEQ ID NOs: 5, 11, 15, 23, 28, 32, 47, 66, 75, 83, 90, 95, 98, 110, 124, 125, 134, 139, 145, 151, 152 or 157 may be modified to comprise a combination of one or more insertions, deletions or transpositions while still maintaining adequate affinity to a toxin produced by C. difficile to have utility in a diagnostic assay.

In various embodiments, the aptamer to a toxin produced by C. difficile includes a sequence of contiguous nucleotides that are identical to a sequence of contiguous nucleotides included in any of the nucleotide sequences shown in Table 4, 6, 8 or 10. In various embodiments, the sequence of contiguous nucleotides in the aptamer to a toxin produced by C. difficile can include any number of nucleotides that are identical to the same number of nucleotides in a sequence of contiguous nucleotides included in any of the sequences shown in Table 4, 6, 8 or 10. In various embodiments, the sequence of contiguous nucleotides in the aptamer to a toxin produced by C. difficile includes a sequence of from about 4 to about 30 contiguous nucleotides that are identical to a sequence of from about 4 to about 30 contiguous nucleotides included in any of the sequences shown in Table 4, 6, 8 or 10. In an exemplary embodiment, the aptamer to a toxin produced by C. difficile includes a sequence of 40 contiguous nucleotides that are identical to a sequence of 40 contiguous nucleotides included in any of the sequences shown in Table 4, 6, 8 or 10 which has 40 or more contiguous nucleotide. In an exemplary embodiment, the aptamer to a toxin produced by C. difficile includes a sequence of 30 contiguous nucleotides that are identical to a sequence of 30 contiguous nucleotides included in any of the sequences shown in Table 4, 6, 8 or 10. In another exemplary embodiment, the aptamer to a toxin produced by C. difficile includes a sequence of 20 contiguous nucleotides that are identical to a sequence of 20 contiguous nucleotides included in any the sequences shown in Table 4, 6, 8 or 10. In yet another exemplary embodiment, the aptamer to a toxin produced by C. difficile includes a sequence of 8 contiguous nucleotides that are identical to a sequence of 8 contiguous nucleotides included in any of the sequences shown in Table 4, 6, 8 or 10. In yet another exemplary embodiment, the aptamer to a toxin produced by C. difficile includes a sequence of 4 contiguous nucleotides that are identical to a sequence of 4 contiguous nucleotides included in any of the sequences shown in Table 4, 6, 8 or 10.

In one embodiment, the aptamer to toxin A is selected from the group consisting of SEQ ID NOS: 1-4, 6-10, 12-14, 16-22, 24-27, or 29-31. In yet another embodiment, the aptamer to toxin A is derived from a consensus sequence selected from any one of SEQ ID NOS: 5, 11, 15, 23 or 28. In one embodiment, the aptamer to toxin A is at least about 95% identical, at least about 90% identical, at least about 85% identical, at least about 80% identical, or at least about 75% identical to any of SEQ ID NOS: 1-31. In another embodiment, the aptamer to toxin A includes a sequence from any of SEQ ID NOS: 1-31 or fragments of any of these.

In one embodiment, the aptamer to toxin B is selected from the group consisting of SEQ ID NOS: 33-46, 48-65, 67-74, 76-82, 84-89, 91-94, 96-97, or 99-108. In yet another embodiment, the aptamer to toxin B is derived from a consensus sequence selected from any one of SEQ ID NOS: 32, 47, 66, 75, 83, 90, 95 and 98. In one embodiment, the aptamer to toxin B is at least about 95% identical, at least about 90% identical, at least about 85% identical, at least about 80% identical, or at least about 75% identical to any of SEQ ID NOS: 32-108. In another embodiment, the aptamer to toxin B includes a sequence from any of SEQ ID NOS: 32-108 or fragments of any of these.

In one embodiment, the aptamer to binary toxin A chain is selected from the group consisting of SEQ ID NOS: 109, 111-123, 126-133, 135-138, 140-144, or 146-150. In yet another embodiment, the aptamer to binary toxin A chain is derived from a consensus sequence selected from any one of SEQ ID NOS: 110, 124-125, 134, 139, or 145. In one embodiment, the aptamer to binary toxin A chain is at least about 95% identical, at least about 90% identical, at least about 85% identical, at least about 80% identical, or at least about 75% identical to any of SEQ ID NOS: 109-150. In another embodiment, the aptamer to binary toxin A chain includes a sequence from any of SEQ ID NOS: 109-150 or fragments of any of these.

In one embodiment, the aptamer to binary toxin B chain is selected from the group consisting of SEQ ID NOS: 153-156, 158-162. In yet another embodiment, the aptamer to binary toxin B chain is derived from the consensus sequence of SEQ ID NOS: 151, 152 or 157. In one embodiment, the aptamer to binary toxin B chain is at least about 95% identical, at least about 90% identical, at least about 85% identical, at least about 80% identical, or at least about 75% identical to any of SEQ ID NOS: 151-162. In another embodiment, the aptamer to binary toxin B chain includes a sequence from any of SEQ ID NOS: 151-162 or fragments of any of these.

The aptamer to a toxin produced by C. difficile can contain any number of nucleotides in addition to the region which binds the C. difficile toxin. In various embodiments, the aptamer can include up to about 100 nucleotides, up to about 95 nucleotides, up to about 90 nucleotides, up to about 85 nucleotides, up to about 80 nucleotides, up to about 75 nucleotides, up to about 70 nucleotides, up to about 65 nucleotides, up to about 60 nucleotides, up to about 55 nucleotides, up to about 50 nucleotides, up to about 45 nucleotides, up to about 40 nucleotides, up to about 35 nucleotides, up to about 30 nucleotides, up to about 25 nucleotides, and up to about 20 nucleotides.

The aptamer to a toxin produced by C. difficile can be selected to have any suitable dissociation constant (K_(d)) for the respective toxin. In an exemplary embodiment, the aptamer to a toxin produced by C. difficile has a dissociation constant (K_(d)) for the respective toxin of about 10 nM or less. In another exemplary embodiment, the aptamer to a toxin produced by C. difficile has a dissociation constant (K_(d)) for the respective toxin of about 15 nM or less. In yet another exemplary embodiment, the aptamer to a toxin produced by C. difficile has a dissociation constant (K_(d)) for the respective toxin of about 20 nM or less. In yet another exemplary embodiment, the aptamer to a toxin produced by C. difficile has a dissociation constant (K_(d)) for the respective toxin of about 25 nM or less. A suitable dissociation constant can be determined with a binding assay using a multi-point titration and fitting the equation y=(max−min)(Protein)/(K_(d)+Protein)+min. It is to be understood that the determination of dissociation constants is highly dependent upon the conditions under which they are measured and thus these numbers may vary significantly with respect to factors such as equilibration time, etc. In other embodiments, the aptamer to a toxin produced by C. difficile is an aptamer with a K_(d) that is less than or equal to the K_(d) of an aptamer selected from the sequences disclosed in Tables 4, 6, 8 and 10.

Since the binary toxin is composed of an A chain and a B chain, more efficient binding may be achieved by using a dimeric or other multimeric form of aptamer. Thus, in another embodiment, the aptamer is a multimerization of any combination of the sequences of Table 8 and the sequences of Table 10. The same strategies could be applied to any aptamer sequence with the appropriate binding characteristics for binary toxin. In another embodiment, an aptamer for A chain could be used in conjunction with an aptamer for B chain to detect binary toxin in a sandwich assay.

Kits Comprising Aptamers to Toxins Produced by C. difficile

The present disclosure provides kits comprising any of the aptamers to toxins produced by C. difficile described herein. Such kits can comprise, for example, (1) at least one aptamer to a toxin produced by C. difficile; and (2) at least one diagnostic testing reagent, such as a solvent or solution. Additional kit components can optionally include, for example: (1) at least one container, vial or similar apparatus for holding and/or mixing the kit components; and (2) apparatus for collecting a sample to be tested for the presence of a C. difficile toxin.

Methods of Detecting Toxins Produced by C. difficile

The present disclosure provides methods of detecting the presence of a C. difficile toxin in a test sample comprising contacting said test sample with an aptamer that binds to a toxin produced by C. difficile wherein said toxin is selected from toxin A, toxin B, binary toxin A chain, and binary toxin B chain. Further disclosed are methods of detecting the presence of a C. difficile toxin comprising aptamers which comprise at least one pyrimidine modified at a C-5 position wherein said at least one pyrimidine modified at a C-5 position comprises a C-5 modification independently chosen from at least one of the modifications shown in FIG. 9. Also disclosed are methods of detecting the presence of a C. difficile toxin comprising aptamers which comprise at least one pyrimidine modified at a C-5 position wherein said at least one pyrimidine modified at a C-5 position comprises a C-5 modification independently chosen from benzylcarboxyamide, naphthylmethylcarboxyamide, tryptaminocarboxyamide, tyrosylcarboxyamide, 2-naphthylmethylcarboxyamide and phenethyl-1-carboxyamide. The methods of detecting a C. difficile toxin disclosed herein may comprise an aptamer which comprises at least one additional chemical modification, wherein said at least one additional chemical modification is a chemical substitution at one or more positions independently selected from the group consisting of a ribose position, a deoxyribose position, a phosphate position, and a base position. Further, the methods of detecting a C. difficile toxin disclosed herein may comprise at least one additional chemical modification is independently selected from the group consisting of a 2′-position sugar modification, a 2′-amino (2′-NH₂), a 2′-fluoro (2′-F), a 2′-O-methyl (2′-OMe) a modification at a cytosine exocyclic amine, a substitution of 5-bromouracil, a substitution of 5-bromodeoxyuridine, a substitution of 5-bromodeoxycytidine, a backbone modification, methylation, a 3′ cap, and a 5′ cap.

The present disclosure provides methods of detecting the presence of a C. difficile toxin in a test sample wherein the method of detecting is selected from a pull-down assay, dot blot assay, PCR assay or sandwich assay.

The present disclosure further provides methods of detecting the presence of C. difficile toxin A in a test sample comprising contacting said test sample with an aptamer comprising a sequence selected from the group consisting of SEQ ID NOS: 1-4, 6-10, 12-14, 16-22, 24-27, or 29-31 or a fragment thereof. Methods of detecting the presence of C. difficile toxin A in a test sample may comprise a pull-down assay, dot blot assay, PCR assay or sandwich assay. Sandwich assays used for detecting the presence of C. difficile toxin A may be selected from an aptamer-target-antibody assay, antibody-target-aptamer assay, and aptamer-target-aptamer assay. Methods of detecting the presence of C. difficile toxin A in a test sample may provide a quantitative measure of toxin A.

The present disclosure further provides methods of detecting the presence of C. difficile toxin B in a test sample comprising contacting said test sample with an aptamer comprising a sequence selected from the group consisting of SEQ ID NOS: 33-46, 48-65, 67-74, 76-82, 84-89, 91-94, 96-97, or 99-108 or a fragment thereof. Methods of detecting the presence of C. difficile toxin B in a test sample may comprise a pull-down assay, dot blot assay, PCR assay or sandwich assay. Sandwich assays used for detecting the presence of C. difficile toxin B may be selected from an aptamer-target-antibody assay, antibody-target-aptamer assay, and aptamer-target-aptamer assay. Methods of detecting the presence of C. difficile toxin B in a test sample may provide a quantitative measure of toxin B.

The present disclosure further provides methods of detecting the presence of C. difficile binary toxin A chain in a test sample comprising contacting said test sample with an aptamer comprising a sequence selected from the group consisting of SEQ ID NOS: 109, 111-123, 126-133, 135-138, 140-144, or 146-150 or a fragment thereof. Methods of detecting the presence of C. difficile binary toxin A chain in a test sample may comprise a pull-down assay, dot blot assay, PCR assay or sandwich assay. Sandwich assays used for detecting the presence of C. difficile binary toxin A chain may be selected from an aptamer-target-antibody assay, antibody-target-aptamer assay, and aptamer-target-aptamer assay. Methods of detecting the presence of C. difficile binary toxin A chain in a test sample may provide a quantitative measure of binary toxin A chain.

The present disclosure further provides methods of detecting the presence of C. difficile binary toxin B chain in a test sample comprising contacting said test sample with an aptamer comprising a sequence selected from the group consisting of SEQ ID NOS: 153-156, 158-162 or a fragment thereof. Methods of detecting the presence of C. difficile binary toxin B chain in a test sample may comprise a pull-down assay, dot blot assay, PCR assay or sandwich assay. Sandwich assays used for detecting the presence of C. difficile binary toxin B chain may be selected from an aptamer-target-antibody assay, antibody-target-aptamer assay, and aptamer-target-aptamer assay. Methods of detecting the presence of C. difficile binary toxin B chain in a test sample may provide a quantitative measure of binary toxin B chain.

The present disclosure further provides methods of detecting the presence of a C. difficile toxin in a test sample comprising contacting said test sample with an aptamer comprising a consensus sequence selected from the group consisting of SEQ ID NOS: 5, 11, 15, 23, 28, 32, 47, 66, 75, 83, 90, 95, 98, 110, 124-125, 134, 139, 145, 151-152 and 157 or a fragment thereof. Methods of detecting the presence of a C. difficile toxin with an aptamer comprising such consensus sequence in a test sample may comprise a pull-down assay, dot blot assay, PCR assay or sandwich assay. Sandwich assays used for detecting the presence of a C. difficile toxin may be selected from an aptamer-target-antibody assay, antibody-target-aptamer assay, and aptamer-target-aptamer assay. Methods of detecting the presence of a C. difficile toxin in a test sample may provide a quantitative measure of such C. difficile toxin.

EXAMPLES

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention as defined by the appended claims. All examples described herein should be considered in the context of standard techniques, which are well known and routine to those of skill in the art. Routine molecular biology techniques can be carried out as described in standard laboratory manuals, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2001).

Example 1 SELEX with C. difficile Toxins A/B and Binary Toxin: Target Procurement

Target Procurement

Targets suitable for SELEX were prepared by PCR amplification of desired gene fragments from C. difficile genomic DNA, cloning in frame into vector pET-51b between the Strep-tag and His-tag sequences, and overexpression in E. coli Rosetta (Table 1). For toxin A, a recombinant polypeptide was obtained that consisted of the carboxy-terminal β-hairpin repeats 17-32. This toxin domain was chosen since a crystal structure has been published for a similar toxin A peptide of just five receptor-binding repeats (Ho, J. G., et al., Proc. Natl. Acad. Sci. USA, 2005. 102(51): p. 18373-8). For toxin B, the amino-terminal catalytic domain was purified; a crystal structure of this domain has been obtained (Reinert, D. J., et al., J. Mol. Biol., 2005. 351(5): p. 973-81). For binary toxin, the full-length CdtA subunit but without the predicted signal sequence was produced in recombinant form (crystal structure available (Sundriyal, A., et al., J. Biol. Chem., 2009. 284(42): p. 28713-9)), and a CdtB fragment (amino acid residues 30-207) was produced that represents the so-called activation domain that has presumably been cleaved off the CdtB precursor protein (Perelle, S., et al., Infect. Immun., 1997. 65(4): p. 1402-7).

Cloning and purification of all C. difficile toxins is shown in FIG. 1A-1D, along with available crystal structures. The recombinant, double tagged proteins were purified via affinity chromatography on Ni-NTA agarose and streptactin agarose using standard protocols.

TABLE 1 PCR amplification of C. difficile toxin genes for cloning and overexpression Target protein PCR amplification Amplicon Domain Size (kDa) Gene PCR primers (nucleotides) Protein (UniProt) (residues) (tagged) tcdA tcdA-6 + tcdA-2 6751-8127  Toxin A (TOXA_CLODI) 2250-2709  57.1 tcdB tcdB-5 + tcdB-7    1-1638 Toxin B (TOXB_CLODI)   1-546 68.8 cdtA cdtA-1 + cdtA-2  103-1387 Binary Toxin A (O32738_CLODI)  35-463 54.7 ctdB cdtB-15 + cdtB-16  90-622 Binary Toxin B (O32739_CLODI)  30-207 26.0 SEQ ID NO: Primer Primer Sequence (restriction sites for cloning underlined) 163 tcdA-6 GCGCAAGCTTCTTCAAAATGGATATATTACTATTGAAAG 164 tcdA-2 GCGCGAGCTCCATATATCCCAGGGGCTTTTAC 165 tcdB-5 GCGCAAGCTTATGAGTTTAGTTAATAGAAAACAGTTAG 166 tcdB-7 GCGCGAGCTCCATCTTCACCAAGAGAACCTTC 167 cdtA-1 GCGCAAGCTTCAAGACTTACAAAGCTATAGTG 168 cdtA-2 GCGCGAGCTCCAGGTATCAATGTTGCATCAAC 169  cdtB-15 GCGCAAGCTTCAAACTAGTACAAGTAATC 170  cdtB-15 GCGCGAGCTCGGTCAAAGAAATTGTTATTTGGG

Example 2 SELEX with C. difficile Toxins A/B and Binary Toxin: SELEX and Pool Affinities

SELEX with the purified His-tagged proteins was performed using Dynabeads® (Talon® or His-Tag) partitioning. Libraries of 40mer random sequences were used that contained one of the six modified nucleotides, 5-tyrosylcarboxyamide-dU (TyrdU), 5-benzylcarboxyamide-dU (BndU), 5-naphthylmethylcarboxyamide-dU (NapdU), 5-tryptaminocarboxyamide-dU (TrpdU), 5-(2-naphthylmethyl)carboxyamide (2NapdU), or 5-phenethyl-1-carboxyamide (PEdU) instead of dU. Seven or eight rounds of selection were carried out, and a kinetic challenge with dextrane sulfate was applied in rounds 2-8. The aptamer pools obtained after the last round of SELEX were tested for affinity to their targets in filter binding assays, and the K_(d)'s and plateaus were determined (Table 2).

The aptamer pools obtained after the last round of SELEX were tested for affinity to their targets in filter binding assays, and the K_(d)'s and plateaus were determined (Table 2). For toxin A, the aptamer pool 4943 (TrpdU) had good affinity with a K_(d)=2.42 nM. Pools 4936 (TyrdU) and 4939 (NapdU) were active, with K_(d)'s of 11.5 and 10.8 nM, respectively. Pools 5564 (2NapdU) and 5577 (2NapdU) were also active, with K_(d)'s of 4.63 and 6.40 nM. For toxin A, the aptamer pool 5570 (PEdU) was the best, with a K_(d)=1.61 nM.

For toxin B, aptamer pools TyrdU, BndU, NapdU, TrpdU, 2NapdU and PEdU demonstrated excellent affinities with K_(d)'s in the range of 0.11-1.11 nM.

Binary toxin A chain was also successful in selecting high affinity aptamers and yielded pool 4758 (TrpdU) with K_(d)=0.40 nM; pool 5567 (2NapdU) with K_(d)=0.19 nM; and pool 5574 (PEdU) with K_(d)=0.30 nM. Binary toxin B chain was used to select active pool 5556 (2NapdU), with K_(d)=7.58 nM.

All pools with sufficient affinity (K_(d) of ˜10 nM or below) were cloned and the sequences of at least 48 clones per pool were determined

TABLE 2 SELEX with C. difficile toxins K_(d) Target Library MOD Sample ID Pool (nM) Plateau Toxin A 40N29.14 TyrdU, S247-R8-S1 4936 11.50 42% BndU S247-R8-S9 n/a¹ 70.30 40% NapdU S247-R8-S17 4939 10.80 48% TrpdU S247-R8-S25 4943 2.42 46% 40N32.24 2NapdU S270-R8-S1 5564 4.63 42% PEdU S270-R8-S9 5570 1.61 21% 2NapdU S270-R8-S29 5577 6.40 23% Toxin B 40N29.14 TyrdU, S247-R8-S3 4937 0.57 43% BndU S247-R8-S11 4938 1.11 24% NapdU S247-R8-S19 4940 0.20 44% TrpdU S247-R8-S27 4944 0.24 43% 40N32.24 2NapdU S270-R8-S5 5566 0.11 18% PEdU S270-R8-S13 5573 0.12 50% 2NapdU S270-R8-S30 5578 0.79 16% Binary 40N58.50 TrpdU S239-R7-S39 4758 0.40 50% Toxin 40N32.24 2NapdU S261-R8-S24 5551 0.22 36% A Chain PEdU S261-R8-S32 5555 3.73 33% 2NapdU S270-R8-S6 5567 0.19 45% PEdU S270-R8-S14 5574 0.30 65% 2NapdU S270-R8-S31 5579 0.33 63% Binary 40N32.24 2NapdU S261-R8-S33 5556 7.58 32% Toxin B Chain ¹pool not cloned due to insufficient affinity

Example 3 Toxin A Aptamer Clones

Representative clones from SELEX pools 4936 (TyrdU), 4939 (NapdU), 4943 (TrpdU), 5564 (2NapdU), 5577 (2NapdU), and 5570 (PEdU) were evaluated for affinity to toxin A in filter binding assays. Nearly all clones had good affinity to the recombinant 57.1 kDa toxin A fragment that had been used for selection, however, only some of the clones demonstrated affinity for the native, 308 kDa toxin A. This is not surprising, since some of the epitopes on the smaller, recombinant protein may not be accessible for aptamer binding of the full-length, native toxin.

The affinities (K_(d)'s) and plateaus of the binding curves are shown in Table 3 and the corresponding sequences are listed in Table 4, with the best clones shown in bold.

Clones from Pool 4936 (TyrdU): Clone 4936-4 represented 20% of the sequences in the pool and was the most active with K_(d)=3.8 nM for the recombinant toxin A domain and K_(d)=14.5 nM for native toxin A.

Clones from Pool 4939 (NapdU): Three unrelated sequences were found five times each in this pool. Clone 4939-280 was the most active clone in this pool, with K_(d)=2.34 nM for the recombinant protein and K_(d)=15.3 nM for native toxin A.

Clones from Pool 4943 (TrpdU): Four clones with good (low nanomolar) affinity for native toxin A were found in this pool. The best clone, 4943-51 (K_(d)=1.78 nM for native toxin A), made up 19% of all sequences in this pool. The other three clones had the common motif NANAnnCNNNCnnCnN (SEO ID NO:5, N=TrpdU; n=A, G, C, or TrpdU). Clones 4943-50, 4943-60, and 4943-49 which possessed only 32 nucleotides instead of the usual 40 within the random region (Kd's, 5.60 nM, 4.57 nM, and 7.91 nM for native toxin A respectively) showed good affinity.

Clones from Pool 5564 (2NapdU): The most active clone of this pool was 5564-49 (K_(d)=1.78 nM for native toxin A). Several other clones were also present in this pool, sharing one of the three sequence patterns

NAAAGNAGGN (SEQ ID NO:11) CGGGNCGNGACAGANCGCA (SEQ ID NO:23), respectively (N=2NapdU; R=A or G; M=A or C; K=G or N).

Clones from Pool 5570 (PEdU): The leading clone, 5570-54, with K_(d)=0.12 nM for the recombinant toxin A and K_(d)=6.91 nM for native toxin A, was the most abundant sequence in this pool.

Clones from Pool 5577 (2NapdU): The active clones shared all or part of the pattern NACCGAACGNNnNCAGNCNGA (SEQ ID NO:28, N=2NapdU; n=A, G, C or 2NapdU). These sequences had been selected in a special SELEX, where a competing toxin A aptamer (4943-51) was present in twofold excess over the target protein concentration.

TABLE 3 Affinities of Aptamer Clones from SELEX with C. difficile toxin A. Toxin A (TOXA_CLODI) Affinity to recombinant protein Affinity to mature protein Aptamer (SELEX Target) (native target) Clone ID Aptamer-ID MOD K_(d) (nM) Plateau K_(d) (nM) Plateau 247-8-1-1 4936-1_0 TyrdU 17.0 33% 247-8-1-3 4936-3_0 TyrdU 23.9 27% 247-8-1-4 4936-4_0 TyrdU 3.80 37% 14.5 29% 247-8-1-9 4936-9_0 TyrdU 33.3 11% 247-8-1-13 4936-13_0 TyrdU 25.7 45% 247-8-1-18 4936-18_0 TyrdU 74.5 36% 247-8-1-32 4936-32_0 TyrdU 19.9 40% 247-8-17-195 4939-195_0 NapdU 23.3  9% >100  2% 247-8-17-196 4939-196_0 NapdU 8.63 34% 61.6 37% 247-8-17-194 4939-194_0 NapdU 20.5  8% 247-8-17-280 4939-280_0 NapdU 2.34 28% 15.3 30% 247-8-17-281 4939-281_0 NapdU 1.35 24% >100  0% 247-8-17-209 4939-209_0 NapdU 11.4 27% 202 55% 247-8-17-202 4939-202_0 NapdU 13.2 34% 104 50% 247-8-17-246 4939-246_0 NapdU 12.5 34% 58.4 31% 247-8-25-49 4943-49_0 TrpdU 1.39 49% 7.91 43% 247-8-25-50 4943-50_0 TrpdU 2.81 36% 5.60 54% 247-8-25-51 4943-51_0 TrpdU 1.23 43% 1.78 54% 247-8-25-60 4943-60_0 TrpdU 2.65 46% 4.57 46% 247-8-25-71 4943-71_0 TrpdU 6.26 18% 247-8-25-73 4943-73_0 TrpdU 0.82 13% 247-8-25-91 4943-91_0 TrpdU 0.43 10% 270-8-1-49 5564-49_0 2NapdU 1.13 30% 1.78 11% 270-8-1-50 5564-50_0 2NapdU 6.99 31% >100  0% 270-8-1-52 5564-52_0 2NapdU 1.86 41% 19.30 20% 270-8-1-58 5564-58_0 2NapdU 6.29 43% 46.00 25% 270-8-1-65 5564-65_0 2NapdU 2.80 20% >100  1% 270-8-1-84 5564-84_0 2NapdU 2.87 46% >100  1% 270-8-1-89 5564-89_0 2NapdU 3.40 34% 11.90 17% 270-8-1-66 5564-66_0 2NapdU 7.17 41% >100  2% 270-8-1-161 5564-161_0 2NapdU 2.71 67% 6.15 12% 270-8-9-50 5570-50_0 PEdU 4.87 23% >100  0% 270-8-9-54 5570-54_0 PEdU 0.12 24% 6.91  8% 270-8-29-1 5577-1_0 2NapdU 1.59 48% 4.97 21% 270-8-29-3 5577-3_0 2NapdU 1.73 39% 5.52 22% 270-8-29-12 5577-12_0 2NapdU 6.48 53% 12.90 19%

TABLE 4 C. difficile Toxin A Aptamer Clones. Lead sequences have their “Aptamer ID No.” bolded and consensus sequences are underlined and appear under the label “Sequence Pattern”. Base capital “N” represents the modified nucleotide as indicated for the particular pool (TyrdU, NapdU, TrpdU, 2NapdU, or PEdU). Base lower case “n” in the consensus sequence indicates a variable base within a motif which is selected from A, G, C or the modified nucleotide for the particular pool (TyrdU, NapdU, TrpdU, 2NapU, or PEdU).  IUPAC nucleotide ambiguity codes were used: M = A or C; R = A or G; K = G or N (N representing the pool-specific modified dU), and a cut-off of 90% representation was used to define consensus. SEQ Aptamer ID NO. ID No. Count Pct Clones from Pool 4936 (TyrdU) 1 4936-4 8 20% AANNCCNANCCNANNCANCACNNNCNNAGANNANNCNANG Clones from Pool 4939 (NapdU) 2 4939-196 5  3% ACNNGNAGNAGCCCNNAANNGGGNNGCGNCGGCANNANGG 3 4939-280 5  3% ACNNGNAGNAGCCCNNAANNGGGNNGCGNCGGCANNANGG 4 4939-246 5  3% CNCGNNAGGGNNNANCCAANACCGNGGNGCCNNAACNAAA Clones from Pool 4943 (TrpdU) Sequence Pattern 5            NANAnnCNNNCnnCnN 6 4943-50 6  7%  AGCANNAAANNANAGACNNNCGNCANCGNCCCCCNNCGGN 7 4943-60 6  7% ANCNCNCACANNANAGACNNNCNNCANGGNCCNCCNGAGA 8 4943-49 4  5%  NGCCNAAACCNANAACCNNNCCACGNGNACNN 9 4943-51 15 19% GNANANCAACNCGNCANGGNAGCGNAANNCNAGCNNAGA Clones from Pool 5564 (2NapdU) 10 5564-49 6  7% CCNGACGGGCGAGGNNCCAACNNACNNCCGNCACNANNGC Sequence Pattern 11                          NAAAGNAGGN 12 5564-52 15 17%  CGCANGNGNNCNGANACACNGGCCNAAAGNAGGNCNGNAG 13 5564-57 1  1%       CCGNNCNAANACCAANGGANAAAGNAGGNAGGAGCNCGCA 14 5564-82 1  1% NACCAGANAGNNANAANACGCNGGCNAAAGNAGGNACNAA Sequence Pattern 15                 GNNRNCMKNCNGA 16 5564-89 2  2%        CNAAANGAAGNNGNCAGNCNGACGCCAGNGCGNACCGN 17 5564-54 1  1%       NGCGNACCCGCNGNNGNCAGNCNGAGAGANCGGCNAAGAA 18 5564-59 1  1%  CNAGCNGCANACCCCACGNNGNCAGNCNGAGCGCCCACN 19 5564-164 1  1%         NGCCNGCCCACCNGNNGNCAGNCNGAGAGCNANCCAAACA 20 5564-167 1  1% CANGCCNGCANACCACACGNNGNNAGNCNGAGGGNNAGG 21 5564-67 4  5%   GGNACCNACCCGCAGNGNNANCANNCNGACCGCGACNNNN 22 5564-58 4  5%                 CNGNNANCCGNCNGACACCNACCNACCGGAGNAAGANCC Sequence Pattern 23                      CGGGNCGNGACAGANCGCA 24 5564-161 4  5%                  CGAGCGGGNCGNGACAGANCGCAGAGCGAAGGCNNANCNAC 25 5564-152 2  2% NCNGANGGGCCAACAAANGNCCGGGNCGNGACAGANCGCA Clones from Pool 5570 (PEdU) 26 5570-54 10 23% GCNGAGGCCGNGCNCANNAANNGAACNNAGAANANCCCNA Clones from Pool 5577 (2NapdU) 27 5577-1 4  9% ACCGCNAAAGNAGGNCACGNNCNAANACCCNGGGAGGNGN Sequence Pattern 28            NACCGAACGNNnNCAGNCNGA 29 5577-3 3  7%      GCNGCCNACCGAACGNNGNCAGNCNGAGCGANCGAACNNG 30 5577-12 3  7% AGCCACGNACANACCGAACGNNANCAGNCNGACGCNGNGA 31 5577-16 1  2%    CCGNGCANACCCCCCNGNNGNCAGNCNGACGGCCAGACAC

Example 4 Toxin B Aptamer Clones

The affinities of aptamers for toxin B were generally very good and correlated well between the 68.8 kDa amino-terminal catalytic domain of C. difficile toxin B fragment that had been used in SELEX and the 270 kDa native, full-length toxin B. Aptamer clones with sub-nanomolar K_(d)'s for toxin B were isolated from all of the pools with TyrdU, BndU, NapdU, TrpdU, 2NapdU and PEdU modified nucleotides (Table 5); the sequences of C. difficile toxin B aptamers are listed in Table 6, with the best clones shown in bold.

Clones from Pool 4937 (TyrdU): Alignments of the TyrdU aptamers indicated the presence of two distinct sequence patterns (YNNSSNGAAW (SEQ ID NO: 32), YGAAWN (SEQ ID NO: 47)), (N=TyrdU; W=A or N; S=C or G; Y=C or N), as well as one orphan sequence.

Clones from Pool 4938 (BndU): This pool contained three unrelated sequences, all were present in multiple copies.

Clones from Pool 4940 (NapdU): The most abundant sequences, including four of the leading clones, contained all or part of the pattern KSGANNGGRW (SEQ ID NO: 66) (N=NapdU; R=A or G; W=A or N; S=C or G; K=G or N). In addition, three unrelated orphan sequences were present.

Clones from Pool 4944 (TrpdU): The majority of the sequences contained the pattern NnCYnnnNCNNnAARNMAMSYN (SEQ ID NO: 75); two other sequences shared a different pattern, CNnGnANCNGGAAAN, (SEQ ID NO:83, N=TrpdU; n=A, G, C or TrpdU; M=A or C; R=A or G; W=A or N; S=C or G; Y=C or N), and four orphan sequences were also present.

Clones from Pool 5566 (2NapdU): Pattern AnCnNNNAAGNGAACNNNnAnnnnnnnnnGnGNNnANA (SEQ ID NO:90, N=2NapdU; n=A, G, C or 2NapdU) was found in a couple of clones. This pool contained two additional unrelated sequences in multiple copies.

Clones from Pool 5573 (PEdU): Two patterns were identified, GCCNNNCNNGNNAAACGNCCNNGANGGCAGCGNN (SEQ ID NO:95) and (SEQ ID NO:98, N=PEdU). Six additional, unrelated clones were present.

Clones from Pool 5578 (NapdU): Two active clones were present in multiple copies. These sequences had been selected in a special SELEX, where a competing toxin B aptamer (4940-23) was present in twofold excess over the target protein concentration.

The highest-affinity aptamers were clones with NapdU or TrpdU modified nucleotides. Five aptamers demonstrated very low K_(d)'s of <0.1 nM for native toxin B.

TABLE 5 Affinities of Aptamer Clones from SELEX with C. difficile toxin B. Toxin B (TOXB_CLODI) Affinity to recombinant protein Affinity to mature protein Aptamer (SELEX Target) (native target) Clone ID Aptamer-ID MOD K_(d) (nM) Plateau K_(d) (nM) Plateau 247-8-3-49 4937-49_0 TyrdU 0.19 23% 0.16  6% 247-8-3-50 4937-50_0 TyrdU 0.36 26% 0.27  4% 247-8-3-51 4937-51_0 TyrdU 0.60 31% 1.07 14% 247-8-3-55 4937-55_0 TyrdU 0.15 39% 0.15 15% 247-8-3-57 4937-57_0 TyrdU 0.15 35% 0.12 11% 247-8-3-66 4937-66_0 TyrdU 0.63 34% 0.59 14% 247-8-3-67 4937-67_0 TyrdU 0.11 21% 0.23 16% 247-8-3-81 4937-81_0 TyrdU 0.23 19% 0.28 13% 247-8-3-85 4937-85_0 TyrdU 0.27 32% 0.72 25% 247-8-3-94 4937-94_0 TyrdU 0.27 27% 0.36 12% 247-8-11-1 4938-1_0 BndU 0.84 23% 2.03 20% 247-8-11-6 4938-6_0 BndU 1.02 16% 1.52 17% 247-8-11-17 4938-17_0 BndU 0.36 32% 0.50 16% 247-8-19-1 4940-1_0 NapdU 0.04 24% 0.06 14% 247-8-19-3 4940-3_0 NapdU 1.04 27% 0.67 12% 247-8-19-6 4940-6_0 NapdU 0.05 15% 0.43  7% 247-8-19-8 4940-8_0 NapdU 0.19 33% 0.27 10% 247-8-19-19 4940-19_0 NapdU 0.11 23% 0.14  8% 247-8-19-23 4940-23_0 NapdU 0.07 37% 0.09 13% 247-8-19-27 4940-27_0 NapdU 0.10 34% 0.09 12% 247-8-27-1 4944-1_0 TrpdU 0.20 26% 0.13 10% 247-8-27-4 4944-4_0 TrpdU 0.10 29% 0.16 11% 247-8-27-5 4944-5_0 TrpdU 0.08 33% 0.09 10% 247-8-27-9 4944-9_0 TrpdU 0.18 36% 0.14  9% 247-8-27-11 4944-11_0 TrpdU 0.14 24% 0.22  6% 247-8-27-14 4944-14_0 TrpdU 0.24 20% 0.70  9% 247-8-27-20 4944-20_0 TrpdU 0.12 25% 0.18  7% 247-8-27-30 4944-30_0 TrpdU 0.06 28% 0.08  9% 247-8-27-34 4944-34_0 TrpdU 0.07 35% 0.41 10% 270-8-5-53 5566-53_0 2NapdU 0.02  5% 0.22 15% 270-8-5-74 5566-74_0 2NapdU 0.04  6% 0.25 21% 270-8-5-77 5566-77_0 2NapdU NT NT 0.23  6% 270-8-13-2 5573-2_0 PEdU 0.03 33% 0.11 15% 270-8-13-3 5573-3_0 PEdU 0.04 52% 0.25 12% 270-8-13-4 5573-4_0 PEdU 0.03 54% 0.08 12% 270-8-13-5 5573-5_0 PEdU 0.06 49% 0.55 14% 270-8-13-9 5573-9_0 PEdU 0.01 45% 0.14 13% 270-8-13-11 5573-11_0 PEdU 6.82 36% 2.70  8% 270-8-13-14 5573-14_0 PEdU 0.04 21% 0.33  9% 270-8-13-23 5573-23_0 PEdU 1.49 35% 0.55  9% 270-8-13-24 5573-24_0 PEdU 0.02 33% 0.11 10% 270-8-30-66 5578-66_0 2NapdU 0.98 14% NT NT 270-8-30-73 5578-73_0 2NapdU 1.62 18% NT NT

TABLE 6 C. difficile Toxin B Aptamer Clones. Lead sequences have their “Aptamer ID No.” bolded and consensus sequences are underlined and appear under the label “Sequence Pattern”. Base capital “N” represents the modified nucleotide as indicated for the particular pool (TyrdU, BndU, NapdU, TrpdU, 2NapdU, or PEdU). Base lower case “n” in the consensus sequence indicates a variable base within a motif which is selected from A, G, C or the modified nucleotide for the particular pool (TyrdU, NapdU, TrpdU, 2NapdU, or PEdU). IUPAC nucleotide ambiguity codes were used: M = A or C; R = A or G; W = A or N; S = C or G; Y = C or N; K = G or N (N representing the pool-specific modified dU), and a cut-off of 90% representation was used to define consensus. SEQ Aptamer ID NO. IN No. Count Pct Clones from Pool 4937 (TyrdU) Sequence Pattern 32                               YNNSSNGAAW 33 4937-50 6  8%                  NANNNCCNNAAGGCNNGGNGAAAACCGCNNNNCGGNNGCG 34 4937-51 3  4%        NGGACCACNANCCCNCCCCACNNNNNCGNGAACNNGAGNN 35 4937-53 2  3%         NGGANCACGNANNCCCACNNACCNNCCNGAAANAGCANNN 36 4937-54 4  5%                              ACNNGGNGAAANNCACNNNCNGCCAGCANCNANNCCCGCN 37 4937-56 2  3%        NNGGCACGAAGNANNGACNNNGAANNGCNGAAACANCNNNNCN 38 4937-57 5  6%              NGGACACCNANNACAGNCNNCGNGAAANNGCANNN 39 4937-61 2  3%            GNGCNGCCCANCNANCCNCNCNNANGAANCCGAANNCC 40 4937-63 2  3% NCCANNCCACCGCGGNGCCACAGNANCANGNNNGCNGAAN 41 4937-74 2  3%                    NCCNANCCNCNCNNCGNGAANCCGAANNGCCNACNGCCNN 42 4937-78 2  3%              NCACAAACNANCCGNNCCNNGGNGAANNCNCAANNNCNGGN 43 4937-81 4  5%                   NACNANCACGCNNNNGGNGAANNGCGAANNCCCGGAGGNN 44 4937-86 1  1%                       AGGCGGGNCNNCANANCCCGCAANNGAANGCACGCNNNCC 45 4937-87 2  3%              GNGACCAACNANGNNANCNNCGNGAANCCGAANNGCCGN 46 4937-94 4  5%            CACACNANNCCCNACCANGANNGGNGAAANAGCANNNCN Sequence Pattern 47                               YGAAWN 48 4937-85 1  1%      CNNACNGAANACNNGAGCAACANCCCGCANNGCCGA 49 4937-61 2  3% GNGCNGCCCANCNANCCNCNCNNANGAANCCGAANNCC 50 4937-74 2  3%         NCCNANCCNCNCNNCGNGAANCCGAANNGCCNACNGCCNN 51 4937-87 2  3%   GNGACCAACNANGNNANCNNCGNGAANCCGAANNGCCGN 52 4937-55 12 16%         AACCCNGNANNCCACACCNNGCCGAAANNGANNNCNNGN 53 4937-52 2  3%         ACCANGNANNACCCCNCCNNGCCGAAANCAGANNNCNGG 54 4937-81 4  5%       NACNANCACGCNNNNGGNGAANNGCGAANNCCCGGAGGNN 55 4937-49 4  5%  NCNANNCCCCGAGNCNNGANANCCACGANNGAANNN 56 4937-56 2  3%           NNGGCACGAAGNANNGACNNNGAANNGCNGAAACANCNNNNCN 57 4937-78 2  3%         NCACAAACNANCCGNNCCNNGGNGAANNCNCAANNNCNGGN 58 4937-73 2  3%                   NCNAACCGGNNCGCANNCACANGAAANNAGGAGGACANCG 59 4937-96 1  1%                           GAGCNAANNGAAGCNACAGGACNCNNGGCACGACGGGNNA 60 4937-53 2  3%                       NGGANCACGNANNCCCACNNACCNNCCNGAAANAGCANNN 61 4937-66 4  5%                   GGNCNCANCGACAAANNNGGAANGNGCGAGCACNANNCGN 62 4937-67 1  1%       GGGCNCAGNANCNGCAGAGCCAGNAGGAACNAGACGGNGN (orphan) Clones from Pool 4938 (BndU) 63 4938-1 5 10% NNGGCGCCGNNNGCGGNANGACNCCCNNNNCNNANGGCNG 64 4938-6 4  8% AGNGCNAGCGACNCCGCGGNACNACNNCNCCCNACNAGN 65 4938-17 3  6% NANAAAGANCNNGCCNNNGNAANNCCNCANGACANAAANA Clones from Pool 4940 (NapdU) Sequence Pattern 66                     KSGANNGGRW 67 4940-1 9 20%              NCCNNNGCGAANCGGGANNGGANNACGGNNGGGCAANAGN 68 4940-23 4  9%            AGGCNCAANGGNGNANCGANNGGAAAGCAGNNAANCGAN 69 4940-19 2  5%              GCGCNCAGNNGGNNGGANNGGGAGNNGGAANNAGGNAGCA 70 4940-31 1  2%  NGGGNCNCAAGNNGGNNGGCCCANNGGGANNGGAAGNCCN 71 4940-6 2  5% CCCNGCGCNGANNNGCAANNAGCACGGCNGNCGGNGAACN 72 4940-3 1  2% NCCANCGGGACCACNAACGNNAGCNCCAGGCGGGACNGNC (orphan) 73 4940-8 1  2% NANCAGACCNCCANCGCGNCACNNANGAGNNGAACACGA (orphan) 74 4940-27 1  2% NANNNGNCCCANNCCCACNNAANGCNAGCACACGNNAACA (orphan) Clones from Pool 4944 (TrpdU) Sequence Pattern 75                        NnCYnnnNCNNnAARWNMAMSYN 76 4944-1 2  4%                     CANGNCNCAANCNNNAAGANAACGNNGACCGCGAGNACCG 77 4944-13 4  9%        NGCNGACAGACACANGNCCCCNCNCNNAAAGANAACGNNG 78 4944-40 1  2%                       ANCACCCCNCNNNAAGANAACGNNCCGGACCGCGCGANAA 79 4944-14 4  9%                      NCNGCNANGNCNNNAAGANCAACCNAAGAGANGCANGANA 80 4944-23 1  2% GNNGGAGCGNNGNGGCNNCACCNNACNGGANCNNGAACCNC 81 4944-11 5 11% GNCGANCNNCAAANNANGNACGANNGACCNAACANGGNAC 82 4944-34 10 22% NGGNNAGCACNNCANNCANGGACCANANAACNCNAGNNNAA Sequence Pattern 83                          CNnGnANCNGGAAAN 84 4944-17 2  4%   ACNNNNCGCACCCGGCCNNANGCCNNGCANCNGGAAANGG 85 4944-4 1  2% NNNNCGGAAGCCGCNNANCCGCCCACNCGGANCNGGAAAN 86 4944-5 1  2% NGNCGAGNAAACGGCGACCGNNNCCCCNGNAGNAACNACA (orphan) 87 4944-9 1  2% NGNNNCAACNANGAANCCAGCNACCGNGCAACCAANGNA (orphan) 88 4944-30 1  2% AGNGNAANAGNAACCCNNAGACNANGCCCNNGGGNANCGG (orphan) 89 4944-20 1  2% NGCGGCNGAAGAAGCANGCAAGNCANCGGNCCGNNGGNAN (orphan) Clones from Pool 5566 (2NapdU) Sequence Pattern 90   AnCnNNNAAGNGAACNNNnAnnnnnnnnnGnGNNnANA 91 5566-53 3 7% CCAGCANNNAAGNGAACNNNAAGGAAGGGAGGAGNNCANA 92 5566-90 1 2% AGACCGNNNAAGNGAACNNNCAACGGGANGCGNGNNAANA 93 5566-74 4 9% AGNGGCGNNAANGCANNNAACGAGCACNGAGGCGNNAANA 94 5566-77 3 7% CNNNNNNACCGCNGCANGACNNNAGCGGCAGNCGNGNGNG Clones from Pool 4473 (PEdU) Sequence Pattern 95        GCCNNNCNNGNNAAACGNCCNNGANGGCAGCGNN 96 5573-23 2  5% GAACGN GCCNNNCNNGNNAAACGNCCNNGANGGCAGCGNN 97 5573-25 1  2% AACNCG GCCNNNCNNGNNAAACGNCCNNGANGGCAGCGNN Sequence Pattern 98                      AGNNNGANCCC 99 5573-3 7 16%    NACGGCANNCNGGNGGCAAGNNNGANCCCNCCGAGCCNAN 100 5573-5 2  5% CGANCACANCGCACANNAGNCAGNNNGANCCCANNAANCA 101 5573-2 14 32% NCAGGNNANACCCAGNGNAGGAAAACGNGNACGNNCCGAN 102 5573-4 8 18% AANNNANGNGANCAANNGAGCAGACCGCCANNNGACNNCG 103 5573-14 3  7% GGNGGNGGAAANNGGCAAGNGNANGGNGGNNACGCCGNAN 104 5573-24 2  5% NGCGNCNGANCCGNAAAACCANNNCAAGCNACCANGNNNA 105 5573-9 2  5% CGCCGNNNCCGNCCGGCCACAANNNAAGNACAANNGGAN 106 5573-11 2  5% NGNCCGCCGACCANNNNCNGNANAGCCNCNNGNAANNAGN Clones from Pool 5578 (2NapdU) 107 5578-66 3  7% GAAAGCNNCGNACGNAGNNGNGAGAGGNCNCNGCCCNCNN 108 5578-73 4  9% ANNAAGCNNGNGGCNGGNAGCNGACAGCCAGGGANNCNGA

Example 5 Binary Toxin (A Chain) Aptamer Clones

SELEX with the recombinant binary toxin A chain (CdtA) yielded active aptamers with TrpdU, 2NapdU, and PEdU modified nucleotides (Table 7).

The sequences and common sequence patterns of CdtA aptamers are shown in Table 8.

Cloning of pool 4758 (TrpdU) revealed clone 4758-6 which comprised 18% of the sequences in that pool and showed good affinity (K_(d)=0.86 nM) to CdtA binary toxin.

Twenty sequences from 2NapdU pools were obtained, most of them with subnanomolar affinity, and several sequence patterns shared between these 2NapdU clones were identified: GAANANnNCCGNGAnGNAANGnnANANNS (SEQ ID NO: 110), ANNRGCNnCCNGGCS (SEQ ID NO: 124), WAWNNANNA (SEQ ID NO: 125), and GGANNGCAGGNNCMC (SEQ ID NO: 134) (N=PEdU; n=A, G, C or PEdU; M=A or C; W=A or N; S=C or G; R=A or G).

PEdU pools contained five active aptamers; they were present in multiple copies, and three of the sequences shared the pattern NAAAWGNNN (SEQ ID NO: 145) (N=PEdU; W=A or N).

TABLE 7 Affinities of Aptamer Clones from SELEX with C. difficile binary toxin A chain. Binary Toxin A Chain Affinity to recombinant CdtA (CdtA_CLODI) Aptamer protein (SELEX Target) Clone ID Seq-ID MOD K_(d) (nM) Plateau 239-7-39-6 4758-6_0 TrpdU 0.86 18% 261-8-24-49 5551-49_0 2NapdU 0.31  9% 261-8-24-50 5551-50_0 2NapdU 0.09 30% 261-8-24-52 5551-52_0 2NapdU 0.14 26% 261-8-24-60 5551-60_0 2NapdU 5.79 60% 261-8-24-81 5551-81_0 2NapdU 0.54 32% 261-8-32-6 5555-6_0 PEdU 0.62 21% 261-8-32-15 5555-15_0 PEdU 1.72 18% 261-8-32-39 5555-39_0 PEdU 0.34 12% 270-8-6-1 5567-1_0 2NapdU 0.15 24% 270-8-6-2 5567-2_0 2NapdU 6.81 25% 270-8-6-10 5567-10_0 2NapdU 0.03 17% 270-8-6-13 5567-13_0 2NapdU 0.03 22% 270-8-6-18 5567-18_0 2NapdU 0.16 10% 270-8-6-34 5567-34_0 2NapdU 0.09 14% 270-8-6-46 5567-46_0 2NapdU 0.05 12% 270-8-14-49 5574-49_0 PEdU 0.16 57% 270-8-14-56 5574-56_0 PEdU 2.92 55% 270-8-31-5 5579-5_0 2NapdU 2.24 13% 270-8-31-7 5579-7_0 2NapdU 2.44 44% 270-8-31-8 5579-8_0 2NapdU 0.53 41% 270-8-31-10 5579-10_0 2NapdU 0.15 36% 270-8-31-11 5579-11_0 2NapdU 0.07 33% 270-8-31-12 5579-12_0 2NapdU 0.97 53% 270-8-31-21 5579-21_0 2NapdU 0.24 35%

TABLE 8 C. difficile Binary Toxin A Chain Aptamer Clones. Lead sequences have their “Aptamer ID No.” bolded and consensus sequences are underlined and appear under the label “Sequence Pattern”. Base capital “N” represents the modified nucleotide as indicated for the particular pool (TrpdU, 2NapdU, or PEdU). Base lower case “n” in the consensus sequence indicates a variable base within a motif which is selected from A, G, C or the modified base within nucleotide for the particular pool (TrpdU, 2NapdU, or PEdU). IUPAC nucleotide ambiguity codes were used: M = A or C; R = A or G; W = A or N; S = C or G; (N representing the pool- specific modified dU), and a cut-off of 90% representation was used to define consensus. SEQ Aptamer ID NO. ID No. Count Pct Clones from Pool 4758 (TrpdU) 109 4758-6 8 18%  GAAGACTTTAATTCTGACATGGTGTCCAATGGCGCGCGAG Clones from Pool 5551, 5567, 5579 (2NapdU) Sequence Pattern 110             GAANANnNCCGNGAnGNAANGnnANANNS 111 5567-30 1  1%             GAANCNGNCCGNGACGNAANG AANANNC 112 5579-21 5  4%             GAANCNGNCCGNGACGNAANGCCANANNCGGAGGGGAN 113 5579-28 1  1%             GAANCNGNCCGNGAAGNAANGCCANANNCGCANG 114 5567-1 11  9%             GAANANGNCCGNGAAGNAANGCGANANNC 115 5567-41  1%             GAANANGNCCGNGAAGNAANGGCANANNCGNCCACGNGGG 116 5551-77 1  1%     CGGGNCACCGCANNCNCCGNGACGNAANGACANANNCGGN 117 5551-60 2  2%    AACCCCGCGGCAANNANCCGNGAAGNAANG AANANNCCGA 118 5579-48 1  1%        ACAGAGGCANNCNCCGNGANGNAANGCAANANNCCGCCGN 119 5567-2 4  3%            NGCAACNANCCGNGANGNAANGCAANANNGCAACANGNGC 120 5567-26 1  1%           GGACNACNCNCCGNGANGNAANGCGAAANNCCCAGANGNA 121 5551-81 4  3% NCGAANGANAACANGNAACNCCGNGANNACANCGAANAGN 122 5579-7 7  6%  CNAAGCNCCGAGGCNNACNCCGNGANCGCANGGNNNAACC 123 5579-12 4  9%    NCGAGCAACGAGNAACNCCGNGANNACAANCGANAGANGA Sequence Pattern 124,125            ANNRGCN CCNGGCS WAWNNANNA 126 5579-11 10 11%  NNGCNACCCAANNAGCN CCNGGCGG GNNAANNANNAGACA 127 5551-64 1  1%      CANCCAANNAGCNCCCNGGCGA NGNAANNANNANGGCACN 128 5551-78 1  1% NCGNANACCGAANNAGCNGCCNGGCGA CCNAANNANNACA 129 5551-50 2  2%    CCNGCCNCANNAGCN CCNGGCGCCCNNAANNANNAAAACN 130 5579-45 1  1%   GACCNCANCANNGGCN CCNGGCCG GNNAANNANNACCACC 131 5567-13 3  2%     NAGAGAAANNGGCNGCGNGGCCACCCNAANNANNAGAGCA 132 5567-34 3  2%   CNCAAGGCNANNGGCN GCNGGCAGA NNAANNANNAAAGNC 133 5567-10 8  6%            ANNGGCN CCNGGCCGGANAANNNANNACCCAGNGAGNGAA Clones from Pools 5551, 5567, 5579 (2NapdU) Sequence Pattern 134                        GGANNGCAGGNNCMC 135 5579-10 2  2%         NAGNCACGGNGAACNGGANNGCAGGNNCCCCCCNGGCNA 136 5551-52 13 15%                GGNCAGCNGGANNGCAGGNNCCCCCCNGANAGGACGGNNN 137 5551-59 2  2%                  GNAGNCGGANNGCAGGNNCCCACCAAACACCNNNGGNAGA 138 5567-46 2  2% CNGGAGACNGGNCAGAACAGCCGGGANNGCAGGNNCACGG Sequence Pattern 139      GAANNGNNCCG 140 5579-5 3  3%   GNNGAANNGNNCCGCCGCCNNNCNGNCCGCGGGNNGCNGN 141 5579-34 1  1% NGNCAGAANNGNNCCGANAGGGNNGCNGCCACNGANAN 142 5551-49 5  6% GCCNNNNGGCGAGGNGAGNNNNCCCAGNCNGANGAAGCNN 143 5579-8 5  6% CGGAGCCCGAAGGNNAAGCGGNNCACCANNANACGANACG 144 5567-18 2  2% CNCCGNANNGCGNCCNGGGCAGNNAANCNANNAGAAGCCA Clones from Pools 5555, 5574 (PEdU) Sequence Pattern 145                           NAAAWGNNN 146 5555-6 9 14% GNGNGNCAGCGCANNANACGCGNAANNAAANGNNNAGAGA 147 5555-15 8 12%               GCGNGNCNGNANNAAAAGNNNGCGGAGGGGNNCCCGGNAC 148 5574-49 13 20%                  NNNCGAGAANAAANGNNNGANACANNACNNANAANANGGN 149 5555-39 3  5% AGCCGGNGNGNGNANNAACNCNNNCGGCNNNCCNCCCGCA 150 5574-56 11 17% CNNGNGNAAACCGNGCGNNAGNANNGGAGANAGCNGACAN

Example 6 Binary Toxin (B Chain) Aptamer Clones

SELEX with recombinant binary toxin B chain (CdtB) yielded active aptamers with 2NapdU modified nucleotides (Table 9). The sequences and common sequence patterns of CdtB aptamers are shown in Table 10. The most active clone, 5556-51, contained all of these patterns, NNARASCS (SEQ ID NO: 151), NNNGGCNNNACG (SEQ ID NO: 152), and AGCCNNNGRCNN (SEQ ID NO: 157) (N=2NapdU; R=A or G; S=C or G), some of which were present in other sequences. Three additional clones had unrelated sequences.

TABLE 9 Affinities of Aptamer Clones from SELEX with C. difficile binary toxin B chain. Binary Toxin B Chain Affinity to recombinant CdtB (CdtA_CLODI) Aptamer protein (SELEX Target) Clone ID Seq-ID MOD K_(d) (nM) Plateau 261-8-33-51 5556-51_0 2NapdU 1.68 38% 261-8-33-57 5556-57_0 2NapdU 11.60 37% 261-8-33-60 5556-60_0 2NapdU 12.30 48% 261-8-33-67 5556-67_0 2NapdU 2.16 45% 261-8-33-83 5556-83_0 2NapdU 7.62 33%

TABLE 10 C. difficile Binary Toxin B Chain Aptamer Clones. Lead sequences have their “Aptamer ID No.” bolded and consensus sequences are underlined and appear under the label “Sequence Pattern”. Base capital “N” represents 2NapdU. IUPAC nucleotide ambiguity codes were used: R = A or G; S = C or G; and a cut-off of 90% representation was used to define consensus. Clones from Pool 5556 (2NapdU) SEQ Aptamer ID NO. ID No. Count Pct Sequence Pattern 151                     NNARASCS 152 NNNGGCNNNACG 153 5556-51 5 15%                  AAGNNAAACCGAGACGCGGCCGGAAGCCNNNGGCNNNACG 154 5556-87 1  3%                    GNNAAACCCCGGGGGGGCCAAGCGCANNNGGCNNNACGAA 155 5556-94 1  3%            CAACGNNAANNAGAGCCNNNGNCCNAACAAANNACGCANG 156 5556-69 1  3% AANCGGAGCCCNNANAACCCNNAAACCCNNNANACCAANN 157                         AGCCNNNGRCNN 158 5556-51 5 15% AAGNNAAACCGAGACGCGGCCGGAAGCCNNNGGCNNNACG 159 5556-60 1  3%                GNNAANNAGAGCCNNNGACNNGAACAGGNNCACGCANNAC 160 5556-57 4 12% CNNGACNGNACCNNNNNCGACACAGAACAGCAAGACCNNC 161 5556-67 2  6% GGACCGANGAANCNAGCNNGNNAANAGCGNNGAGCNANCC 162 5556-83 2  6% CACNNAGCAACCGACACAAGNNGNNCCGNNANCCGNNANA

Example 7 Use of Aptamers for Toxin A/B and Binary Toxin as Diagnostic Reagents: Pull-Down Assays

Aptamers were used to specifically pull-down their respective targets, toxin A, toxin B, or binary toxin, from spiked samples, thus affinity purification of these proteins is achieved, as needed.

In this assay, biotinylated aptamers were immobilized on MyOne streptavidin beads and mixed with their targets for 1 h to allow binding. The beads were then washed, and captured target was tagged with NHS-Alexa-647. After extensive washing, the captured targets were eluted with 20 mM NaOH, neutralized, analyzed by SDS-PAGE, and proteins were visualized using the cy5 channel.

FIG. 2 illustrates the following pull down assay results: the toxin A aptamer pulled down toxin A (recombinant or native) with good specificity over the control proteins toxin B or BSA; the toxin B aptamer pulled down toxin B (recombinant or native), but not toxin A; the aptamer for binary toxin A subunit pulled down CdtA, but not CdtB; and no proteins were present in the pull-down fraction when a random aptamer was used.

Example 8 Use of Aptamers for Toxin A/B and Binary Toxin as Diagnostic Reagents: Dot Blot Assays

Aptamers were used for the detection of toxins in dot blot assays, e.g. using biotinylated aptamers and a signal amplifying enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP).

Toxin detection was demonstrated in simple dot blots of toxin A and B (FIGS. 3A and 3B). In this assay, 1 μL of serially diluted target was spotted and air-dried onto nitrocellulose membranes. After blocking, individual biotinylated aptamers were added (1 nM), followed by SA-AP conjugate (200 ng/mL) and developed with NBT/BCIP substrate. Detection limits were ˜1 fmole (300 pg) for either toxin A and B with aptamers. Monoclonal antibodies used for the same assay and at the same concentration (1 nM) were not as good, but were able to detect 2 fmoles (600 pg) of toxin A and 20 fMoles (6 ng) of toxin B.

Example 9 Use of Aptamers for Toxin A/B and Binary Toxin as Diagnostic Reagents: Catch 1 & 2 Assay with qPCR Detection

A Catch 1-Catch 2 assay is illustrated at FIG. 8. Toxins A and B were detected quantitatively via qPCR of catch 2 eluates (FIG. 4A-B). In catch 1, spiked samples containing toxin (0.001-10 nM) and excess BSA (1.5 μM) were equilibrated with aptamers comprising a photocleavable biotin-D spacer (10 nM) and captured on streptavidin agarose beads (relatively clear beads which allow light to pass through). After wash removal of free protein, the toxin (target) of the catch 1 samples was tagged with NHS-biotin, aptamers were photocleaved off the streptavidin agarose beads and complexes were captured on MyOne streptavidin beads (catch 2). Since after photocleavage the photocleavable biotin-D spacer could no longer mediate binding to the MyOne streptavidin beads, in catch 2 the binding to the MyOne streptavidin beads was mediated by the NHS-biotin on the toxin (target). After wash removal of free aptamers, the target-bound aptamers were eluted at high pH and used for qPCR; standard curves for the aptamers were run side-by-side. Quantitative results for toxin A and B were obtained at concentrations of >0.1 nM only. There was non-specific background at the lower target concentrations, and pPCR curves reached a plateau after less than 12 cycles. This was most likely due to considerable carry-over of free aptamers during catch 1 and 2, and due to a rather high (10 nM) aptamer concentration.

Example 10 Use of Aptamers for Toxin A/B and Binary Toxin as Diagnostic Reagents: Aptamer-Target-Antibody Sandwich Assay

Toxins were detected in streptavidin plate sandwich assay, using biotinylated aptamers and monoclonal antibodies (FIG. 5A-B).

Toxin detection was demonstrated for toxins A and B. Biotinylated aptamers (1 pmole/well) were immobilized on a streptavidin plate, and target proteins were added (1 nM, 100 pM, 10 pM, no protein), corresponding to 100 fMoles (30 ng), 10 fMoles (3 ng), 1 fMole (300 pg), and no protein control. The plates were washed and monoclonal antibodies to Toxin A or B (2 nM each) were added and allowed to bind for 1 h with shaking at RT. Complexes were detected with goat-anti-mouse HRP conjugate and TMB as HRP substrate (FIGS. 5A and 5B).

The sandwich assay produced robust results with target concentration-dependent signals and low background. All four toxin A aptamers were able to detect 10 μM toxin A (1 fMole, 300 pg) regardless of their K_(d) and did not cross-react with toxin B. Aptamers for toxin B, in spite of better K_(d)'s, had poor sensitivity in this assay, possibly due to overlapping binding sites of the aptamer and antibody. One of the toxin B aptamers (4937-49) did cross-react with toxin A, which is consistent with data from pull-down experiments.

Example 11 Use of Aptamers for Toxin A/B and Binary Toxin as Diagnostic Reagents: Antibody-Target-Aptamer Sandwich Assay

Aptamers were used for the detection of toxins in antibody-aptamer sandwich assays, e.g. in a dipstick-type assay format, using biotinylated aptamers and monoclonal antibodies (FIG. 6).

Monoclonal antibodies to toxin A and B were spotted separately on a small (0.6 cm×2.5 cm) strip of nitrocellulose and air-dried. After blocking with SB18T+1% BSA, the strips were placed upright in a deep well plate and 0.6 mL samples containing toxin A and/or toxin B or controls were added. After shaking for 1 h at RT, the strips were washed 3×. Biotinylated aptamers (1 nM) were added (0.6 mL) and allowed to bind for 1 h at RT. The strips were washed again and developed with 1 nM streptavidin-alkaline phosphatase conjugate (1 h) and NBT/BCIP (FIG. 6).

The toxin A aptamer 4943-51 detected toxin A accurately in all samples containing only toxin A or both toxins A and B (1000 fMoles or 100 fMoles), and did not cross-react with toxin B. Similarly, toxin B aptamers were able to detect toxin B. Background was high especially in the toxin B spot, even when no protein was present in the sample and when random control aptamers were used, suggesting non-specific binding of the streptavidin-alkaline phosphatase conjugate to the toxin B monoclonal antibody.

Example 12 Use of Aptamers for Toxin A/B and Binary Toxin as Diagnostic Reagents: Aptamer-Target-Aptamer Sandwich Assay

Aptamers were used for the detection of toxins in aptamer-aptamer sandwich assays, e.g. in a bead-based assay format using a pair of aptamers, without the need of any antibodies (FIG. 7).

Capture beads were prepared by attaching the first, biotinylated aptamer (clone 4758-6) to MyOne™ streptavidin beads. A sample containing target protein (CdtA) in serial dilutions was added, and CdtA was allowed to bind to these capture beads. After washing the beads, a second, radiolabeled CdtA aptamer clone was added for equilibrium binding. Then the mixture was filtered through MAHVN plates (0.22μ), using the capture beads themselves for partitioning. This method will detect only labeled aptamers that are bound in sandwich-type format to the streptavdin-aptamer 4758-6-CdtA complex.

The results of the capture bead assay for CdtA aptamer pairs are shown in FIG. 7. Suitable pairs of aptamers produced target concentration-dependent signals. No signals were produced if the same aptamer (binding to the same epitope) or a control aptamer (binding to a different target) was used.

This assay can be used to screen aptamers for binding to distinct epitopes on the target, that is, each aptamer binding to a separate site, as opposed to competing for the same epitope. 

What is claimed is:
 1. A method of detecting the presence of C. difficle toxin in a test sample comprising contacting said test sample with an aptamer that binds to C. difficile toxin B wherein said aptamer comprises a sequence selected from the group consisting of SEQ ID NOS: 67-71.
 2. The method of claim 1, wherein said aptamer further comprises at least one addition chemical modification, wherein said at least one addition chemical modification is a chemical substitution at one or more position independently selected from the group consisting of a ribose position, a deoxyribose position, a phosphate position, and a base position.
 3. The method of claim 2, wherein said at least one additional chemical modification is independently selected from the group consisting of a 2′-amino (2′-NH₂), a 2′-fluoro (2′-F), a 2′-O-methyl (2′-Ome), a modification at a cytosine exocyclic amine, a substitution of 5-bromouracil, a substitution of 5-bromodeoxyuridine, a substitution of 5-bromodeoxycytidine, a backbone modification, methylation, a 3′ cap, and a 5′ cap.
 4. The method of claim 1, wherein said aptamer comprises a K_(d) for C. difficile toxin B of 30 nM or less.
 5. The method of claim 1, wherein said aptamer is nuclease resistant.
 6. The method claim 1, wherein said aptamer has a slow off rate. 